Crohn's disease is a chronic granulomatous ileocolitis, of unknown etiology, which generally affects the patient during the prime of life. Medical treatment is supportive at best and patients afflicted with this disorder generally live with chronic pain, in and out of hospitals, throughout their lives. The disease bears the name of the investigator that convincingly distinguished this disease from intestinal tuberculosis in 1932. This distinction was not universally accepted and the notion of a mycobacterial etiology has never been fully dismissed. Nevertheless, it was 46 years after the distinction of Crohn's disease and intestinal tuberculosis before research attempting to reassociate mycobacteria and Crohn's disease was published. Recently, there has been a surge of interest in the possible association of mycobacteria and Crohn's disease due largely to the isolation of genetically identical pathogenic M. paratuberculosis from several patients with Crohn's disease in the United States, the Netherlands, Australia, and France. These pathogenic organisms have been isolated from only a few patients and direct evidence for their involvement in the disease process is not clear; however, M. paratuberculosis is an obligate intracellular organism and strict pathogen which strongly suggests some etiologic role. Immunologic evidence of a mycobacterial etiology, as assessed by humoral immune determinations, have been conflicting, but evaluation of the more relevant cellular immunity have not been performed. Data from histochemical searches for mycobacteria in Crohn's disease tissues have been equally conflicting with acid-fast bacilli detected in 0 to 35% of patients. Animal model studies have demonstrated the pathogenic potential of isolates as well as elucidating the complexity of mycobacteria-intestinal interactions. Treatment of Crohn's disease patients with anti-mycobacterial agent has not been fully assessed, although case reports suggest efficacy. The similarities in the pathology, epidemiology, and chemotherapy of Crohn's disease and the mycobacterioses are discussed. The issue is froth with controversy and the data generated on the association of mycobacteria and Crohn's disease are in their infantile stages such that a general conclusion of the
legitimacy of this association cannot be made. While no firm evidence clearly implicates mycobacteria as an etiologic agent of Crohn's disease, the notion is supported by suggestive and circumstantial evidence, and a remarkable similarities to other known mycobacterial diseases.

The disease that Crohn, Ginzberg, and Oppenheimer described in 1932 was a chronic low-grade inflammation of the terminal ileum (59). Earlier cases may have been documented (196, 298), but the authors failed to receive recognition for describing a new disorder. These earlier cases were called nonspecific granulomata and were sorted from those that had previously been termed hyperplastic tuberculosis of the intestine. Although Crohn's disease was first described as a segmental disease of the small intestine, in 1960, it was recognized that the same disorder affected the colon and had been confused with ulcerative colitis (161). In recent years the lesions of Crohn's disease have been recognized in the mouth, larynx, esophagus, stomach, skin, muscle, synovial tissue, and bone (17, 141, 142, 152, 171, 182, 206, 297). Thus, Crohn's disease may be considered a newly recognized disease, with a defined clinical and pathologic description dating back only to the 1960's. Although the terms Crohn's disease, Crohn's colitis, Crohn's ileitis, and regional ileitis have been with us longer, there is uncertainty as to the accuracy of these diagnoses prior to 1960. To this date, Crohn's disease and ulcerative colitis continue to be confused clinically and the term inflammatory bowel disease (IBD) was developed to comprise both diseases.

Patients afflicted with this disorder generally suffer with chronic weight loss, abdominal pain, diarrhea or constipation (obstruction), vomiting, and generalized malaise. Between 70 to 80% of Crohn's disease patients require surgical resection of the diseased intestine (83, 102, 120, 243). Difficulties usually are not ended by surgical intervention, and most patients will suffer recurrences and require further surgical procedures (71, 109, 123, 161, 181, 291). Generally, patients live with chronic pain, in and out of hospitals, throughout their lives. Mortality is approximately 6% (83, 239). Perhaps more important than mortality, is the quality of life of Crohn's disease patients. Less than 50% of patients consider their quality of life to be "good"; suboptimal psychosocial function is recorded in 30-54% of patients (83, 230, 291). These patients with inflammatory bowel disease (an estimated 2 million, 200,000 whom are children, in the United States alone), represent a very unhappy population with little prospect for relief. The etiology remains obscure and medical treatment is supportive at best (102, 238, 243). Twenty-five years after the original description, Crohn and Yarnis wrote (61): "From this small beginning we have witnessed the evolution of a Frankenstein monster that, if not threatening to life, frequently results in serious illness, often prolonged and debilitating."

In their original disease description, Crohn and co-workers (59) attempted unsuccessfully to infect guinea pigs, rabbits, and chickens with diseased intestine and lymph nodes. Van Patter, W. (Ph.D. thesis, University of Minnesota, 1952) inoculated 131 animals, including guinea pigs, rabbits, cats, rats, and chickens with diseased tissues from 43 patients; all remained normal. Mitchell and Rees (189, 190) and others (42, 43, 270) claimed to have transmitted a granuloma-inducing agent to the footpads of mice by inoculation of diseased tissue filtrates. Others have reported that they transmitted ileitis to rabbits by intraserosal inoculation of tissue filtrates (258). These studies and others could not be reproduced and did not withstand reexamination. The results could not be confirmed by others; granulomas were sometimes produced with filtrates from control tissues, and many of the granulomas were found to contain foreign material such as bone, hair, and synthetic fibers (5, 26, 240, 304). In a multicenter study in which various investigators exchanged material, a transmissible histologic abnormality could not be reproduced (304). In recent years, Das et al (69) has described the production of lymphomas or
plasma cell hyperplasia in nude mice by the injection of Crohn's disease tissue. Additionally, these authors claim that sera from Crohn's patients react with certain cells or cell-types in these murine lymphomas as demonstrated by the fluorescent antibody technique (13, 299, 310). This quite fascinating, and unexplained, phenomenon is currently under investigation and still awaits judgment from other laboratories. Thus, over the years, definitive evidence of an infectious (or at least transmissible) agent has not been forthcoming.

Nevertheless, despite the lack of any clear-cut evidence of an infectious etiology, investigators continue to seek agents in diseased tissues, and every few years, a new putative agent emerges. This continued pursuit probably is related to the lack of acceptance of other theories, including a role for allergy, autoimmune disease, and dietary factors. Additionally, the pathology of the disease, the multiple familial occurrences, and the multiple remote lesion sites all suggest an infectious process. Viruses (10, 96, 104, 108, 222, 223, 293, 294, 306) and L-form bacteria (19, 139, 261, 294) have been incriminated most commonly, but in recent years, these all have been discarded as candidate agents. To this date, the etiology of Crohn's disease, being infectious, transmissible, or not, has eluded the scientific community. Now, over 55 years since its distinction from tuberculosis, attention has been driven back to the beginning: "Is Crohn's disease a mycobacterial disease after all?" (97).

Medical historians suggest that Crohn's disease may first have been described as early as 1682-1771, or even earlier (143). Reports of diseases suggestive of Crohn's disease have appeared in 1806, 1813, 1828, 1875, 1907, 1908, 1909, and 1913 (143). Whether these cases actually were Crohn's disease, will remain unknown. Mycobacteria were not discovered until 1874 when Armauer Hansen described acid-fast bacilli in leprosy patients (118). The organism causing tuberculosis, which would be confused with Crohn's disease for years to come, was not discovered until 1882 (149), and intestinal tuberculosis was not recognized until several years later. Nevertheless, a disease was described in the early 1900's that was similar to intestinal tuberculosis, but acid-fast organisms could not be demonstrated. Dalziel (64) in 1913 described several patients with chronic intestinal enteritis which, although very similar to intestinal tuberculosis, was believed to be a new disorder. He drew attention to a recently described disease in cattle called pseudotuberculosis (now known as paratuberculosis) "in which the histological characters and naked-eye appearances are as similar as may be to those we have found in man". Dalziel goes on to state: "In my cases the absence of acid-fast bacilli would suggest a clear distinction, but the histological characters are so similar as to justify a proposition that the diseases may be the same". Also in 1913, contrary to the views of Dalziel, Ignard (cited by 197) wrote that "In many cases of hyperplastic tuberculosis of the intestine, no tubercles, giant cells or bacilli are found. The lesion is consisted of a mixture of variable proportions of tuberculous and inflammatory elements. In certain cases, the last only exists. Nevertheless, these inflammatory tumors should be classified among the tuberculous". The view of Ignard predominated, and these unusual intestinal diseases became known as hyperplastic tuberculosis.

By the 1920's, the belief was fading that intestinal tuberculosis occurred without acid-fast bacilli or caseous necrosis, and a disease known as "non-specific granulomata" emerged (197, 298). In these cases, as well as those described by Crohn et al (59) in later years, the authors each discussed "the remarkable resemblance" of these cases to intestinal tuberculosis. The landmark article by Crohn, Ginzberg, and Oppenheimer (59) recognized regional ileitis as a separate and unique disease entity and displaced the long-held belief of a mycobacterial etiology. We now know that hypertrophic intestinal tuberculosis (3) and tuberculosis without caseation or demonstrable acid-fast bacilli (296) do exist, as well as a distinct disease known as Crohn's disease. Nevertheless, over the years, the notion recurs that Crohn's disease might in fact be mycobacterial in origin.

Attempts to isolate mycobacteria from Crohn's disease patients dates back before the time that this disease was recognized as a distinct entity. During these periods, as discussed, cases of intestinal tuberculosis were documented in which the tubercle bacillus could not be visualized or isolated from diseased tissues. Even after the recognition of Crohn's disease, investigators continued to seek mycobacteria in diseased specimens without success. These attempts were made almost exclusively with Lowenstein-Jensen (LJ) media and seeking Mycobacterium tuberculosis almost exclusively. As knowledge was gained about the cultivation of mycobacteria, investigators came to realize that routine methods and media were not appropriate for all mycobacteria and many species failed to grow under these conditions. Because the clinical and pathological similarities between Crohn's disease and intestinal tuberculosis continued to suggest some form of relationship, researchers adapted different bacteriological methods and immunologic tests in attempts to find an elusive Mycobacterium species responsible for Crohn's disease.

Perhaps the first concerted effort to isolate mycobacteria from Crohn's disease patients was presented by Van Patter, W. (Ph.D. thesis, University of Minnesota, 1952). In these studies, Van Patter reported the results of 1,762 cultures from 43 patients with Crohn's disease. By employing 7 different types of media and
incubation periods up to 15 months for some cultures, he isolated acid-fast organisms from 3 patients (7%) after 6, 7.5, and 8 months incubation. These organisms could not be subcultured and were never formally identified.

For the next 25 years, not a single report appeared on the attempt to isolate mycobacteria from Crohn's disease patients. Although undoubtedly attempts were made over the years, such data were presented only as laboratory information related to a case report in order to rule out the possibility of intestinal tuberculosis. In 1962, Golde and McGill (101) addressed the issue of atypical mycobacteria (more appropriately called mycobacteria other than tuberculosis or MOTT) in Crohn's disease and suggested searching for these organisms rather than for M. tuberculosis alone. The ability of these organisms to produce chronic intestinal disease and the similarities of Crohn's disease to tuberculosis and Johne's disease (paratuberculosis) were also noted in their report. It was another 16 years before an original article appeared implicating mycobacteria as etiologic agents in Crohn's disease.

In 1978, a revival of the notion that mycobacteria might be related to Crohn's disease occurred with the articles by Burnham et al (33, 34). These authors described the isolation of M. kansasii from the lymph node of a single patient with Crohn's disease and pleomorphic material, suggestive of cell wall deficient (CWD) organisms, from 22 of 27 Crohn's disease patients, 7 of 13 ulcerative colitis patients, and 1 of 11 controls. It was proposed that CWD-forms of M. kansasii played an etiologic role in both Crohn's disease and ulcerative colitis, however, this theory held a short life.

The most damaging evidence to the theory of M. kansasii as an etiologic agent in Crohn's disease was the failure of Burnham et al (33, 34) to identify the CWD forms and their assumption that they were forms of M. kansasii. This organism is recognized as an opportunistic pathogen causing disease predominantly in individuals with underlying chronic disease (105, 215, 244, 284). It is not a primary pathogen in healthy individuals, and is generally nonpathogenic in animals; a few strains may be pathogenic for mice. Although the natural reservoir of M. kansasii is not known (it is not found in soil or dust), it has been isolated from a variety of water sources (122, 241) and healthy animal tissues including lymph nodes (135, 302). Thus, M. kansasii was not a good candidate for consideration as a primary pathogen. The pleomorphic organisms, however, continued to be investigated. Stanford, J. L. (Proc. 2nd Intl. Workshop on Crohn's Disease, Martinus Nijhoff Publ., 1981, pp. 274-277) cultured patient lymph nodes on many bacteriological media, in addition to LJ and Robertson's cooked meat medium, and reported the isolation of irregular acid-fast masses from 42 of 76 patients with Crohn's disease, 14 of 27 with ulcerative colitis, and 3 of 41 control lymph nodes. Although these masses resembled CWD forms, they could not be identified. In an attempt to indirectly support the notion that these masses were CWD mycobacteria, Stanford chemically induced CWD forms of M. kansasii, filtered them through 0.45 and 0.22 mcm filters, and then inoculated the filtrates onto culture media. After a period of time, abnormal acid-fast forms appeared which were visually indistinguishable from those observed in IBD tissues. Attempts to isolate classical mycobacteria from these experimentally induced acid-fast masses were unsuccessful. In an accompanying paper, White (White, S. A., Proc. 2nd Intl. Workshop on Crohn's Disease, Martinus Nijhoff Publ., 1981, pp. 278-282) presented additional data suggesting that this acid-fast material was of mycobacterial or corynebacterial origin. An examination of culture material by serology and thin-layer chromatography revealed that most material containing these coryneform bacteria or acid-fast forms reacted with highest titers to M. kansasii-related mycobacteria and Corynebacterium antisera. Additionally, White found evidence of mycolic acids in some of the acid-fast masses. From their data, Stanford and White concluded that IBD may be associated with or caused by an organism from the Mycobacterium-Corynebacterium axis.

In 1984 there was again a surge of activity on the role of mycobacteria and Crohn's disease, and yet a different Mycobacterium species. From this period on, there have been more reports on mycobacteria and Crohn's disease than in the last 50 years, and as expected, the data are equally conflicting and controversial. In 1984, Chiodini et al published a series of papers describing the isolation of 2 strains of an M. paratuberculosis-like organism from 11 patients with Crohn's disease but not from 3 with ulcerative colitis or 3 with other bowel diseases (49). Detailed techniques and characteristics of the isolates (48) as well as antimicrobial susceptibility profiles (50) were reported in addition to animal susceptibility studies. The isolates were pathogenic for mice by the intravenous or intraperitoneal route, but not for chickens, guinea pigs, rats, or rabbits. Oral inoculation of one of the strains into a newborn goat produced a granulomatous ileocolitis without observable acid-fast bacilli after 5 months. Immunologic studies on this animal failed to show seroconversion, except for an early IgM response which rapidly subsided. Although the authors presented data suggesting skin test reactivity in this animal to M. paratuberculosis PPD but not M. tuberculosis PPD, the level of observed reactivity would not be considered positive in a clinical setting.

The authors concluded that their isolates were strains of M. paratuberculosis or a biovariant of that species and suggested that this organism plays an etiologic role in at least some cases of Crohn's disease. An editorial that accompanied some of these papers (97) suggested that these studies "provide the most intriguing evidence yet generated regarding a possible cause of this important illness" and that "scientists have come closer than ever to fulfilling Koch's postulates and developing a test system for Crohn's disease". On the other hand, this editorial also recognized some of the pitfalls of these studies, the need for further research, and that skepticism would and should exist.

Shortly thereafter, Chiodini et al reported that primary isolation of their putative agent occurred in a CWD form or spheroplast (55). On primary culture these organisms appeared as non-acid fast coccobacillary forms that had the ultrastructural appearance of spheroplasts, and after several months incubation transformed into characteristic M. paratuberculosis-like organisms. Employing genetic techniques, i.e., restriction polymorphism of the 5s ribosomal RNA genes, spheroplasts were found to be identical to the parent bacillary M. paratuberculosis-like forms (55), and were identified as strains of M. paratuberculosis. Additionally, these workers isolated spheroplasts, four of which transformed into M. paratuberculosis, from 16 of 26 patients with Crohn's disease (61%), but not from 13 patients with ulcerative colitis or from 13 with other bowel disorders. Although these spheroplasts remain unidentified, 7 of 10 tested seroagglutinated with specific M. paratuberculosis antisera suggesting that these unidentified forms were also M. paratuberculosis. Some of these CWD forms required up to 1-1/2 years incubation for primary emergence of colonies. The authors indicated that the presence of CWD forms could account for i) the inability to demonstrate acid-fast bacilli in patients' tissues; ii) the failure to demonstrate a strong and consistent immunologic response because CWD mycobacteria are generally of low immunogenicity; and iii) the previous failure to isolate these organisms because of the caustic nature of most other techniques for processing mycobacteriology specimens. Based on available information about mycobacterial spheroplasts, which suggests that only bacillary forms are pathogenic, they postulated that a very slow rate of reversion with subsequent local hypersensitivity-type immunologic responses could account for the chronicity of Crohn's disease.

In a series of studies the organisms were definitively identified as strains of M. paratuberculosis. By both restriction polymorphism of ribosomal 5s genes (R. J. Chiodini and T. J. Yang, Abstr. Annu. Meet. Am. Soc. Microbiol. 1986, U-20, p.122; R. J. Chiodini, Proc. 21st Joint U.S.-Japan Leprosy Tuberc. Conf. 1986, pp. 8-12; R. J. Chiodini, Proc. 22nd Joint U.S.- Japan Tuberc. Conf. 1987, pp.47-51) and studies of random gene sequences (175, 176), restriction patterns were found to be identical between the Crohn's disease isolates and M. paratuberculosis. These papers not only served to confirm the identification of these isolates, but also described the first genetic technique capable of separating the pathogenic M. paratuberculosis from its close relatives of the environmental M. avium-M. intracellulare (MAI) complex. Previously applied techniques, i.e., DNA:DNA hybridization, failed to separate this closely related group of organisms (174, 307). Although taxonomically it has been proposed to classify these related organisms into a M. avium-M. intracellulare-M. paratuberculosis complex (R. J. Chiodini, Proc. 22nd Joint U.S.-Japan Tuberc. Conf., 1987, pp. 47-51), sufficient genetic divergence is present to maintain M. paratuberculosis as a distinct species.

At a research conference on paratuberculosis in Melbourne in 1986, Coloe et al (Coloe, P. J., C. R. Wilks, D. Lightfoot, and F. A. Tosolini. Aust. Microbiol. 7:188, 1986) reported on the isolation of M. paratuberculosis from one of 30 patients with Crohn's disease. The organism was isolated from colonic material after 16 weeks incubation; cultures of the draining lymph nodes were negative. This isolate was identified by biochemical criteria and cellular fatty acid profiles. This work represented the first confirmation of the isolation of M. paratuberculosis from a Crohn's disease patient. At present, Coloe et al have cultured biopsies from approximately 50 Crohn's disease and 50 control (ulcerative colitis, normal tissue) patients. Although some acid-fast growth has been observed on some cultures from Crohn's disease patients, these slow growing isolates have not yet been characterized (P. J. Coloe, personal communication, 1988).

Whitehead examined the serologic activity of sera from the Crohn's disease patient from whom this Australian isolate was obtained, as well as a few additional patients (Whitehead, J. Proc. 2nd Intl. Coloq. Paratuberc., 1988, in press). Employing an immunoblot technique with patient sera on M. paratuberculosis antigens separated by polyacrylamide gel electrophoresis, this investigator found that identical antigen bands were recognized by the sera from the Crohn's disease patients examined and cattle naturally infected with M. paratuberculosis.

Graham and co-workers in 1987 reported their results of culture and DNA hybridization (106). They described the isolation of mycobacteria from 47.6% of 105 specimens including those from Crohn's disease, ulcerative colitis, and control patients. Mycobacteria were isolated from 9 of 59 patients with Crohn's disease, 9 of 19 with ulcerative colitis, and 18 of 27 non-IBD controls. Most isolates were of the MAI complex and M. fortuitum complex, with a single M. kansasii isolate and one they claimed to be similar to M. paratuberculosis isolated from an ulcerative colitis patient (genetic studies conducted after publication suggested that this isolate was not M. paratuberculosis). They did not find any specific association between mycobacteria and Crohn's disease and brought to light the widespread occurrence of mycobacteria in diseased tissues. Yoshimura et al (307) failed to find any association between mycobacteria and Crohn's disease by the use of DNA:DNA hybridization methods. Application of this technique to 31 biopsy specimens revealed mycobacteria-related sequences in 10 of 19 patients with Crohn's disease, 2 of 6 with ulcerative colitis, and 1 of 6 controls. Again their results did not support the notion that mycobacteria are etiologically related to Crohn's disease. The authors recognized that their findings did not rule out mycobacteria as etiologic factors in Crohn's disease, but the results may have been limited by low sensitivity of the methods employed (42% positive rate is equal to the sensitivity they achieved by cultural methods). In their article, Yoshimura et al (307) presented additional data supporting the identification of the Crohn's disease-isolated mycobacteria previously reported (48) as M. paratuberculosis.

The culture results of Graham and co-workers (106) provide some useful and important information and illustrate the ubiquitousness of some Mycobacterium spp. These workers applied tissue processing techniques of lower stringency than that recommended for the isolation of M. paratuberculosis, and these methods probably account for their results. Although the authors suggest that their method using 0.1% hexadecylpyridinium choride (HPC) is that recommended by the National Animal Disease Center (NADC), this laboratory actually recommends 0.1% benzalkonium chloride (279) or more recently, 0.75% HPC (187). At concentrations less than 0.75% HPC, contaminants, which include environmental mycobacteria, commonly overgrow cultures from clinical specimens. Since organisms of the MAI and M. fortuitum complex are widespread in the environment, processing techniques of low stringency would result in the isolation of these species from a variety of sources. The results of Graham et al (106) are comparable to those obtained from environmental sources. MAI complex can be isolated from 26-63% of soil samples, 50% of tap water samples, 13% of dust samples, and 35% of air samples (27, 95, 122, 241, 289). M. fortuitum can be found in 39% of soil samples, 63% of dust samples and 25% of air samples (137, 241, 284).

An interesting feature of the culture results of Graham et al (106) is the specimen type from which mycobacteria were isolated. Except for a single strain of M. fortuitum complex, mycobacteria could not be isolated from resected tissues; but 35 strains of mycobacteria (predominantly MAI and M. fortuitum) were isolated from biopsy specimens of aphthous ulcers. These ulcers provide a suitable micro-environment for the propagation of such environmental organisms. On the other hand, Graham et al (106) isolated as yet unidentified spheroplasts primarily from resected tissues of Crohn's disease patients rather than aphthous ulcers. Retrospectively, these culture data appear to support, rather than refute, a CWD mycobacterial etiology. Thus, as in all other diseases, the area from which material is obtained for culture is of great importance, as are the techniques applied to tissue processing.

At about the same time, Haga (115) briefly reported that he was unable to isolate mycobacteria from 17 fecal specimens, 5 bowel resection, or 9 biopsy specimens from Crohn's disease patients; although, he did report the isolation of acid-fast coccoid bodies from a Crohn's disease patient which could not be identified or subcultured. S. R. Pattyn, F. Portaels, and Y. Van Maercke presented their culture results at the meeting of the International Working Group on Mycobacterial Taxonomy (IWGMT) held in Bithoven, The Netherlands in September of 1987 (S. R. Pattyn, personal communication) and later published their data in the form of a letter (L. J. Colemont, S. R. Pattyn, P. P. Mitchielsen, J. H. Pen, P. A. Pelckmans, Y. M. Van Maercke, and F. Portaels. Lancet 1:294-295, 1988). These workers examined tissues from 32 patients with Crohn's disease and demonstrated acid fast bacilli in 11 (34%) by acid-fast staining. Cultivation attempts yielded 2 strains of M. chelonei which were said to be mycobactin-dependent; mycobacteria could not be isolated from the remaining 9 cases in which acid-fast bacilli were observed. The authors acknowledged that their processing technique, i.e., 0.15% benzalkonium chloride and 0.5% NaOH, may have been too deleterious for recovery of other acid-fast bacilli; M. paratuberculosis is known not to survive exposure to NaOH decontamination.

In 1986 and 1987 a few additional reports on mycobacteria and Crohn's disease appeared, but these were not research papers. Tytgat and Mulder (278) presented a review on the etiology of Crohn's disease and were the first to report, in other than abstract form, the isolation of M. paratuberculosis from a patient in the Netherlands. Data published in this review represented the first independent duplication of previous efforts and confirmation that M. paratuberculosis may be isolated from some cases of Crohn's disease. While all the active theories on the etiology of Crohn's disease were addressed, the authors considered that "a microbial aetiology, particularly mycobacterial, seems the most promising".

Further data from some of the previously mentioned investigators were recently presented at an international research symposium sponsored by the National Foundation for Ileitis and Colitis, Inc. late in 1987.

Haagsma et al presented data on cultivation of mycobacteria from patients with Crohn's disease (113) and the presence of M. paratuberculosis antibodies in Crohn's disease patients (114). They cultured 66 surgical specimens and isolated M. paratuberculosis from 1, M. fortuitum from 1, and acid-fast material from 2. Colonies of M. paratuberculosis emerged after 11 and 16 months incubation on Herrold's egg yolk and Ogawa media, respectively. These studies, however, had two major flaws: the processing protocol changed sometime during the study and control tissues were not cultured. Their isolates of M. paratuberculosis were found to be genetically identical to those isolated by Chiodini in the United States (56). These investigators have recently isolated an additional strain of M. paratuberculosis from a Crohn's disease patient, bringing the number of Crohn's disease-associated M. paratuberculosis isolates to 2 out of 88 specimens examined (Haagsma, J., personal communication, 1988).

Yoshimura et al (308) presented data on characterization of some of their previous isolates (106). Their isolates were predominantly of the MAI complex, probably serovar 19, but some remain unidentified by biochemical or genetic methods. Many of their isolates could not be fully characterized due to their slow growth, but of those examined, none were M. paratuberculosis.

G. Gitnick, et al (98) described their efforts to isolate mycobacteria from resected Crohn's disease tissues and inoculation of animals with their organisms. These authors cultured tissues from 27 patients with Crohn's disease, 29 with ulcerative colitis, and 26 with other bowel diseases. Three strains of mycobacteria were isolated, of which two remain uncharacterized. One isolate from a patient with Crohn's disease was identified as M. chelonei, another was said to be similar to M. paratuberculosis, and the third isolate from a cancer patient remains uncharacterized. The two isolates from Crohn's disease required 3 and 12 months incubation, respectively. Acid-fast spherules were isolated from a few Crohn's disease patients as well as controls. The M. chelonei isolate was inoculated orally into newborn goats which subsequently developed a transient diarrhea. Three animals died 5-10 days post-inoculation. Intestinal lesions were limited to mild inflammation and colonic infiltration with polymorphonuclear cells. Animals receiving the uncharacterized M. paratuberculosis-like organism remained clinically and pathologically normal. Of interest is the apparent acute diarrheal disease produced by M. chelonei since this organism is generally associated with immunocompromised hosts or with traumatic wounds (31, 110, 111, 205, 263). An acute intestinal disorder produced by M. chelonei could have significant meaning to both the veterinary and medical professions. However, the authors did not adequately rule out other neonatal diseases of goats as a possible cause of the observed diarrhea and acute bowel inflammation. The latest data from this laboratory indicates that they have isolated mycobacteria from 3 out of 27 patients with Crohn's disease, 1 out of 31 ulcerative colitis patients, and 1 out of 27 controls. Two of the 3 isolates from Crohn's disease are M. paratuberculosis (one genetically confirmed); the other is the strain of M. chelonei reported above. The strains from controls and an ulcerative colitis patient are slow-growers, as of yet unidentified, but do not appear to be M. paratuberculosis (Gitnik, G., personal communication, 1988).

Lastly, at the American Gastroenterological Association meeting in May 1988, the abstracts related to mycobacteria and Crohn's disease were only genetic studies on some of the isolates. McFadden et al (McFadden, J. J., J. Thompson, E. Green, S. J. Hampson, J. Sranford, J. Haagsma, R. Chiodini, and J. Hermon-Taylor. Gastroenterol. 94:A294, 1988) presented data showing that the M. paratuberculosis organisms isolated by Chiodini et al (48-50, 55), a M. paratuberculosis organism isolated independently in the Netherlands (113), a M. paratuberculosis strain isolated from primates (172), and wild-type M. paratuberculosis associated with disease in ruminants were all identical. Additionally, they found that spheroplasts isolated from Crohn's disease and other patients by Stanford (266) were not M. paratuberculosis but a heterologous group of organisms. Hampson et al (Hampson, S. J. J. McFadden, J. Thompson, E. Green, M. Moss, F. Portaels, and J. Hermon-Taylor. Gastroenterol. 94:A170, 1988) used DNA probes to examine mycobacteria isolated from AIDS patients, patients with atypical mycobacteriosis, and healthy individuals. They used a specific M. paratuberculosis DNA probe and were unable to identify this species in any of their material. They concluded that the absence of M. paratuberculosis in their study population confirms that this organism has only been isolated from Johne's disease
and Crohn's disease, two pathologically similar disease processes.

Lastly, the Bovine Pathology Laboratory of the Lyon Veterinary School in France isolated a strain of M. paratuberculosis from a 45-year old woman with Crohn's disease. This isolate was identified by numerical taxonomic methods at the Laboratoire Central de Recherches Veterinaires. Drs. Descos and Perard of the Lyon-Suds Hospital and Lyon Veterinary School, respectively, have initiated a study to attempt isolation from fecal and biopsy specimens from approximately 50 patients with Crohn's disease.

It is now clear that a host of different mycobacteria can be isolated from Crohn's disease patients, as well as control populations, and that diseased tissue may be a suitable micro-environment for colonization of some of these species (Table 1). Most of these organisms are environmental opportunists (Table 2), although a few investigators have isolated the pathogenic M. paratuberculosis (Table 3). Unfortunately, in all studies reported to date, different methods have been used (Table 4) and as would be expected, many different results have been obtained. This is true not only for the cultural studies, but for immunological studies as well. Thus, no consistent data are available that support the role of mycobacteria in Crohn's disease.

This controversy is perhaps heightened by the evidence that M. paratuberculosis may be an etiologic agent in Crohn's disease because the organism itself is controversial. M. paratuberculosis has never been subjected to numerical taxonomic methods and has been ignored by the IWGMT. This organism is the slowest growing of the culturable mycobacteria and has a variety of sensitive growth requirements for cultivation (51-54). It generally takes years to become fully proficient at working with this species. Often even the most experienced mycobacteriologists have difficulty growing it because conventional methods are not appropriate. In addition some laboratory and other strains of M. paratuberculosis that are being used for study are actually MAI. The lack of any previous suggestion that M. paratuberculosis had public health significance has also added to its disregard by medical mycobacteriologists. These workers are now studying M. paratuberculosis but lack the background to cope successfully with the peculiarities of this species. The experience and expertise is in the hands of veterinary mycobacteriologists who generally do not have access to human tissue. It is interesting to note that all investigators who have been successful in isolating M. paratuberculosis from Crohn's disease patients were trained originally in veterinary mycobacteriology and had years of experience dealing with this peculiar species.

The microbiologic data generated on mycobacteria and Crohn's disease is rather similar to that observed in leprosy. Although leprosy is caused by M. leprae, a host of other mycobacteria have been associated with the lesions. Such mycobacteria, which are termed leprosy-associated mycobacteria (LAM) or armadillo-derived mycobacteria (ADM), can be isolated from leprosy skin lesions of humans and armadillos (72, 74, 200, 224-226). Isolation rates in armadillos average about 50% for naturally infected, experimentally infected, and non-infected animals. Organisms isolated include MAI, M. scrofulaceum, M. gordonae, M. terrae, and several groups of unclassified, difficult to grow mycobacteria (72, 226). Draper (74) suggested that infection with M. leprae favored the multiplication of environmental and other culturable mycobacteria within the lesions. These organisms are considered by most to be "insignificant" or "contaminants" (74, 226).

In addition to LAM or ADM, leprosy lesions are associated with large numbers of coryneform bacteria, termed leprosy-derived corynebacteria (LDC) (58, 136, 232, 234). Although it was once thought that these organisms were non-acid fast forms of M. leprae, it is now known that they are not related to mycobacteria and probably play a role similar to the LAM (74, 232). Unlike the LAM, LDC are primarily associated with the lesions and are rarely isolated from non-infected tissues. Some investigators believe that the LDC and LAM have a symbiotic relationship with M. leprae, while others believe these organisms represent opportunistic superinfection of the leprosy lesion. Regardless of which view is correct, it is clear that the LAM and LDC have no significance to the disease, their isolation in culture should be disregarded and they should be considered contaminants. It is also relevant to point out that even with the large numbers of M. leprae, LAM, and LDC present in leprous tissues, many cultivation attempts are negative.

A similar phenomenon occurring in Crohn's disease would clearly account for the numerous environmental mycobacteria isolated from Crohn's disease and control tissues, and also account for the coryneform bacteria isolated from Crohn's disease patients (S. White and J. Stanford (Proc. 2nd Intl. Workshop on Crohn's disease, Martinus Nijhoff Publ., 1981). Because > 106 viable bacteria are required to yield a single colony when some mycobacteria are subcultured in vitro (presumably more bacteria are needed for primary culture) (224), low numbers of a pathogenic strain or species overgrown by environmental mycobacteria and coryneform bacteria would be difficult to isolate particularly if the organism has peculiar in vitro growth requirements. In addition, organisms present in numbers < 106/gram tissue are not detectable by acid-fast staining and light microscopy. If Crohn's disease patients are infected with low numbers of M. paratuberculosis or some other Mycobacterium species and super-infected with organisms similar to the LAM and LDC, such a situation could account for the data generated.

The use of immunologic responsiveness to specific antigens is a well recognized method of determining the etiology of infectious disease. Generally these determinations are based on the demonstration of rising antibody titers, but in some diseases, particularly chronic conditions, such is often not demonstrable. Diagnostic assays of chronic disease are therefore more generally based on cell-mediated immunity (CMI) or delayed-type hypersensitivity (DTH) rather than humoral responses. Despite the appropriate evaluation of cellular immunity in chronic conditions, such as Crohn's disease, most studies to date have examined humoral immunity and these have been quite limited. Except for a few scattered reports, immunologic studies related to mycobacteria and Crohn's disease have been conducted either in direct response to bacteriologic data (presented under Cultural Evidence), or involved the use of mycobacterial antigens in accessing general immunologic functions.

Morganroth and Watson (195) examined delayed cutaneous reactions and precipitating antibodies in Crohn's disease patients to antigens of atypical mycobacteria of the Runyon groups I, II, and III, as well as standard purified protein derivative (PPD). No increased incidence of sensitivity to these antigens was detected in 22 Crohn's disease patients as compared to controls. Unfortunately, the authors did not describe the species of mycobacteria examined or the nature of the antigens used. Thayer et al (272) examined skin test reactivity of Crohn's disease patients to tuberculin PPD, in addition to several other non-mycobacterial antigens, to evaluate anergy in Crohn's disease. These authors also failed to find an increased skin reaction to PPD in Crohn's disease patients and found no evidence of anergy as assessed by skin test reactivity. Bird and Britton (23) also failed to find increased responses to M. tuberculosis in Crohn's disease patients by the
lymphocyte blastogenesis assay. Matthews et al (168) examined sera from 24 Crohn's disease patients in an agglutination assay with antigens from M. paratuberculosis and M. avium, in addition to antigens from non-mycobacterial microbes. As antigen, these authors used phenol-killed whole cells of the mycobacteria which displayed wide cross-reactivity. The majority of sera from Crohn's disease patients (79 to 96%) agglutinated M. paratuberculosis and M. avium cells, but such reactivity was also observed in an equal number of controls. Thus, there was no clear difference observed between Crohn's disease patients and controls.

In conjuction with their isolation of M. kansasii in culture, Burnham et al (33, 34) determined that in skin tests with antigens prepared from M. kansasii a high proportion of Crohn's disease patients showed an increased response as as compared to controls. No differences in reactivity between controls and Crohn's disease patients were noted with antigens prepared from 16 other Mycobacterium species. White et al (292) in Burnham's group also found increased reactivity of Crohn's disease patients to M. kansasii antigens. By the use of an indirect fluorescent antibody technique, positive responses were found in 9 of 11 sera from patients with Crohn's disease but not in any of 33 control sera. Based on these and their culture data, these authors suggested CWD M. kansasii as an etiologic agent in Crohn's disease. During the same year, however, Whorwell et al (294) reported their inability to demonstrate M. kansasii in tissues by immunofluorescence, and by 1980, members of Burnham's group reported that they were unable to duplicate their original immunologic findings. Although increased responsiveness to skin tests with M. kansasii antigens was stll observed in Crohn's disease patients was, they also found increased responsiveness in their control population (77). Also in 1980, Grange et al (107) reported increased IgA and IgM antibodies to M. tuberculosis in patients with Crohn's disease. They noted that responses of tuberculosis patients were predominantly of the IgG class rather than IgA and IgM as found in Crohn's disease.

In conjunction with the isolation studies reported by Chiodini et al (48,49), these authors (273) presented data suggesting increased serologic reactivity to M. paratuberculosis antigens in Crohn's disease by the ELISA technique. Patients with Crohn's disease had a statistically significant increase in antibody titer to a protoplasmic antigen of M. paratuberculosis as compared to controls. Examining cross-reactivity between antigens, these authors found 52.5% and 39% cross reactivity of their antigen with M. kansasii and M. tuberculosis, respectively. As a result of this cross-reactivity, a significant proportion of Crohn's disease patients' sera also reacted to M. kansasii antigens. These results have not been duplicated in any other laboratory.

Cho et al (57), examined seroreactivity of Crohn's disease and control patients to common mycobacterial antigens and a species-specific peptidoglycolipid of M. paratuberculosis. Increased reactivity of Crohn's disease patients in comparison to controls was not observed with either antigen. They concluded that, as in paratuberculosis, seroreactivity is not a reliable method of examining the relationship between Crohn's disease and mycobacteria. Haga et al (116) also failed to duplicate the results of Thayer et al (273) and reported that the antibody titers to M. paratuberculosis of 32 Crohn's disease patients, 37 ulcerative colitis patients, or 48 non-IBD controls did not differ in any immunoglobin class.

Cho et al (57) recognized that these inconsistencies in seroreactivity did not necessarily contradict the on-going theories, particularly when organisms related to the MAI complex were involved. The MAI group is so widespread that all individuals, healthy and diseased, are likely to be exposed to these organisms and their antigens. Seroreactivity of the general population to MAI antigens is expected, not only because of their ubiquitous nature, but also because antigens in the Order Actinomycetales are highly conserved. Common antigens, particularly those of major cellular components, exist between all Family members of the Order including Streptomycetacae, Nocardiaceae, Actinomycetaceae, Mycobacteriaceae, in addition to Corynebacteriaceae. Thus, when unpurified antigens, such as sonicated whole cells, are used a wide range of reactivity among normal and diseased populations would likely be found. Some studies have shown that 40 to 60% of the general public reacts to MAI antigens, probably related to their constant exposure to these agents in the environment (211, 302). Most of the "common" mycobacterial antigens, such as lipoarabinomannan, are cell wall components which would be lacking in a CWD form. Unless a specific antigen can be located that will not cross-react with MAI and other related organisms, a difference among populations is not likely to be noted. Although Cho's use of a species-specific antigen from M. paratuberculosis (57), was the proper approach, animals naturally infected with M. paratuberculosis do not respond to this antigen and this antigen has not been found in wild-type strains isolated from clinical cases, suggesting that this antigen does not exist or is not expressed in wild-type strains (37). Furthermore, this species-specific antigen was later found not to be species-specific but identical to that of MAI serovar 2 (38) and some recent data suggest that these laboratory strains in which this specific antigen was detected are not M. paratuberculosis but rather MAI (R. J. Chiodini, Proc. 22nd Joint U.S.-Japan Tuberc. Meet., 1987, pp. 47-51).

Jiwa et al (130) described IgG serum antibodies to mycobacterial PPD's in Crohn's disease patients. These investigators examined seroreactivity to PPD's prepared from M. tuberculosis, M. kansasii, M. phlei, M. paratuberculosis, and M. smegmatis and found that Crohn's disease patients have elevated antibody titers to all species examined. Serologic studies conducted with a crude antigen and 3 antigenic fractions of M. paratuberculosis also showed a slight, but insignificant, increased antibody titer in Crohn's disease patients as compared to controls. Such widespread reactivity to PPD, probably based on a ubiquitous cross-reactive antigen, is highly indicative of sensitization by environmental organisms gaining immune access through a defective mucosal barrier.

Kobayashi et al (147) sought antibodies to mycobacteria in Crohn's disease patients by the ELISA method using lipoarabinomannan (LAM) and a protoplasmic antigen preparation of M. paratuberculosis as antigen. These authors failed to find any significant elevation in IgA, IgG, or IgM antibody levels in Crohn's disease as compared to controls. Based on their findings these authors concluded that, since all chronic infections have an associated serologic response to the etiologic agent, their failure to find a response in Crohn's disease greatly diminishes the likelihood of mycobacteria as etiologic agents. This statement is not entirely correct. For example, patients with the tuberculoid form of leprosy, by definition fail to mount a humoral immune response (46, 119, 158, 202). Additionally, although these authors attempted to address and correct errors made in other studies, their results with control antigens did not agree with those previously reported. Crohn's disease patients have a generalized increased antibody response to enteric organisms (11, 25, 86, 154), but in the study by Kobayashi et al (147), antibody titers to lipid A were not increased. The use of LAM as a broad mycobacterial antigen may also be inappropriate because most normal individuals have demonstrable LAM titers, probably related to exposure to environmental mycobacteria. Although patients with mycobacterial diseases such as leprosy and tuberculosis generally have LAM titers higher than control groups, it is unclear if other mycobacterioses produce similar responses. Additionally, this study was the first to attempt duplication of the serologic results of Thayer et al (273) using a similar protoplasmic antigen. Some questions have been raised regarding the nature of their preparation, however, since Kobayashi et al reported that this antigen had at least 20 SDS-PAGE bands while Thayer et al reported that their antigen contains only 5 SDS-PAGE bands (W. R. Thayer, J. A. Coutu, R. J. Chiodini, and H. J. Van Kruiningen. Gastroenterol. 88:1613, 1986).

D. C. Markesich et al (167) have studies the interaction of peripheral blood monocytes with M. paratuberculosis to determine if monocytes from Crohn's disease patients react differently to mycobacteria as compared to controls. These investigators found that macrophages from Crohn's disease patients inhibited growth more efficiently than controls and that the survival of M. paratuberculosis in Crohn's disease monocytes was significantly less than in controls. The authors noted that their study was conducted with a limited number of patients and the possibility or effect of increased activated macrophages in Crohn's disease patients (180, 207) was not assessed.

Das et al (Das, P. K., J. L. G. Blaauwgeers, A. W. Slob, J. Spies, A. Chand, A. Kolk, and H. J. Houthoff. Gastroenterol. 94:A88, 1988) examined the possible relationship of mycobacteria and Crohn's disease by using immunoblot analysis and a lymphoproliferative assay. They found that sera from patients with Crohn's disease reacted with various mycobacterial and gut-associated antigens, and that many sero-reactive epitopes were shared between mycobacteria and human gut tissue. Thus they concluded that the pathogenesis of Crohn's disease could involve either cross-reactive epitopes or idiotypes, without the persistent presence of viable mycobacteria. Their lymphoproliferative assay showed that the lymphocytes from 5 out of 6 patients with Crohn's disease reacted specifically to M. paratuberculosis antigens, whereas those from controls, ulcerative colitis, and bowel cancer patients did not. Other than the brief, inconclusive, report by Thayer et al (Thayer, W.R., J. A. Coutu, R. J. Chiodini, and H. J. Van Kruiningen. Gastroenterol. 90:1662, 1986), these preliminary studies represent the only investigation of CMI responsiveness to mycobacterial antigens in Crohn's disease.

Ajitsu et al (Ajitsu, S., S. Mirabella, and H. Kawanishi. Gastroenterol. 94:A4, 1988) examined the immunologic responsiveness of murine intestinal tissues by studying the response of gut-associated lymphoid tissues to orally administered M. paratuberculosis antigens in young and old mice. They found that cells from old mice responded to M. paratuberculosis antigens to a much greater extent than those from young mice (stimulation index > 10 vs. < 3). Additionally, cells from old mice responded by producing IgG, IgM, and IgA, while cells from young mice produced only low levels of IgA. These authors concluded that the oral tolerance to M. paratuberculosis antigens in old mice is impaired, that the gut mucosal immunity in these older mice is hyper-reactive, and that these age-associated features are due in part to impaired antigen-specific T suppresser cells with over-reactive antigen specific B and T helper cells. Although not suggested by the authors, their observation that young animals respond poorly to oral M. paratuberculosis challenge, as compared to aged mice, could explain why M. paratuberculosis appears only to successfully infect only young animals. The hyper-reactive mucosal immunity in the aged could explain the age-dependent resistance to M. paratuberculosis infection (51).

Crohn's disease patients do not have any consistent, reproducibly significant, antibody response against mycobacterial antigens. Some studies have demonstrated responses in some patients, while others have not. Patients with mycobacterioses usually have a humoral immune response, therefore the lack of such in Crohn's disease could be strong evidence against the etiologic role of mycobacteria in this disease. While some patients with pulmonary tuberculosis (45, 76, 78, 201) and, occasionally, lepromatous leprosy (46, 47, 119, 202) fail to elicit a humoral immune response, these cases are generally associated with bacterial overload and anergy. Immunologic non-responsiveness could also be caused by advancing age (235, 283), debility (78), or malnutrition (78, 158). Such is not the case in Crohn's disease. However, during certain periods in mycobacterioses, a humoral immune response is not demonstrable. Primary immunity to mycobacteria is cell-mediated. Only as the disease progresses, and there is an increasing bacterial load, does the humoral immune response become activated with the production of antibody. Patients with polar tuberculoid leprosy, in which there is a low bacterial load, fail to elicit a humoral response to M. leprae (119, 276). Most cattle with paratuberculosis have a low antibody response, but it is often not greater than that in noninfected animals (51). Cattle with overt clinical disease, unless anergic, do have a significant antibody response as compared to control cattle; variable immune responsiveness occurs in animals during subclinical disease which is perhaps more relevant to Crohn's disease. Clinical paratuberculosis (severe diarrhea and rapid weight loss) is considered the terminal stage of the disease since animals generally die within a few months after clinical onset (51). During this subclinical period, when animals appear normal but suffer subtle decreased productivity, weight loss, and increased susceptibility to other infections (51, 53, 151), immunologic responses are not readily distinguished from non-infected animals (51). The level of immunity in these animals probably is masked by cross-reactive responsiveness to environmental mycobacteria and related taxa (2, 66, 94, 121, 155, 156, 177-179, 220, 233). Only in recent years has the use of purified antigens been successful in diminishing some of the non-specific cross-reactive responses (1, 305), and shown that each animal species, i. e., cattle, sheep, and goats, responds serologically to different antigenic determinants (248, 249). In humans it may be necessary to examine tissue lymphoid cells rather than those of the peripheral blood (82, 134, 191, 192), to seek antigens which may be masked and therefore not demonstrable (252-255), to purify antigens to reduce non-specific reactions due to environmental mycobacteria (2, 66, 94, 121, 155, 156, 177, 178, 220, 233), and/or define antigenic determinants recognized by the human immune system since they may be different from those of ruminants.

At this time, insufficient information is available on the immune response of early paratuberculosis in cattle to judge whether or not a demonstrable immune response is present and how it is elicited. It is also unclear what type of immune response occurs in animals with tuberculoid-type paratuberculosis, in which acid- fast bacilli are not demonstrable but are culturable (32, 51). A tuberculoid response, i.e., DTH reaction to M. paratuberculosis antigens would be expected in these animals, but more often than not, they fail to mount any humoral, cellular, or DTH reactions (51). Likewise, no information is available on the immunologic responses in primary human intestinal tuberculosis. The only report on CMI responses in intestinal tuberculosis (22) does not define the disease as primary or secondary or whether concurrent pulmonary disease was present. Patient histories suggest that those with ulcerative or ulcerotrophic (secondary) tuberculosis generally respond to intradermal injection of tuberculin PPD, unless pulmonary disease is far advanced (anergy). This response would be expected because most patients with pulmonary tuberculosis are PPD reactive. Patients with primary intestinal tuberculosis caused by M. bovis likewise produce a positive response to PPD, but patients with hypertrophic intestinal tuberculosis are generally non-responsive to PPD if the infection is caused by M. tuberculosis, rather than M. bovis (3). Even in cases where M. tuberculosis has been isolated, the patients do not respond to skin tests. Thus, PPD reactivity is of no diagnostic value in primary hypertrophic intestinal tuberculosis if the causative agent is M. tuberculosis (3). The reason(s) for this lack of reactivity are unknown and have not been investigated. It would be important to determine both humoral and CMI responses, in subclinical tuberculoid paratuberculosis and intestinal tuberculosis, particularly the hyperplastic types. Such information on defined mycobacterial disorders would be invaluable for understanding the lack of consistent immunologic reactivity in Crohn's disease if the
etiology is related to a Mycobacterium spp.

Since Crohn's disease is a granulomatous disease, and therefore assumed to be DTH mediated, it would be appropriate to study cell-mediated rather than humoral responses. If an infectious agent is present in Crohn's disease, it is present in low numbers and probably not in sufficient amount to stimulate a humoral immune response. The presence of granulomas in Crohn's disease is evidence of a functional and responsive CMI system, and such responses should be measurable. However, if the agent is similar to M. paratuberculosis, it may be difficult to sort specific responses from cross-reactive responses without using purified antigens or defined antigenic determinants recognized by the human immune system. Although an attempt to demonstrate a CMI or DTH response to mycobacteria in Crohn's disease is the obvious route of investigation, as yet, only limited inconclusive studies have been performed.

The ultimate goal in determining the etiologic relationship of an organism to a disease state is to demonstrate the association of that organism with the lesions. Routine acid-fast staining has not been successful in Crohn's disease; the absence of acid-fast bacilli was a criterion for the classification of Crohn's disease as a distinct disease entity. There have been few reports on the search for mycobacterial antigens or acid-fast bacilli in tissues from patients with Crohn's disease, but no good evidence of tissue-lesion associated mycobacteria has been found.

The first published attempt to identify mycobacteria in tissue sections from patients with Crohn's disease was the report of Whorwell et al (294) in 1978. These investigators sought evidence for the presence of M. kansasii, in addition to other pathogenic microorganisms, by immunofluorescence in tissues from patients with
Crohn's disease and ulcerative colitis. No evidence of infection was found. Haga (115) also reported that he was unable to demonstrate M. paratuberculosis antigens in 18 formalin-fixed tissues from Crohn's disease patients by immunohistochemistry using anti-M. paratuberculosis antisera.

On the other hand, as discussed under cultural data, Yoshimura et al (307) employing liquid genomic DNA:DNA hybridization was able to detect mycobacteria-related sequences in 53% of Crohn's disease patients, 33% of ulcerative colitis patients, and 17% of controls. None of these sequences, however, were identical to M. paratuberculosis, and some question whether or not the methodology was sensitive and specific enough to determine if the sequences were even mycobacterial in origin.

Van Kruiningen et al (282) used peroxidase-anti-peroxidase immunohistochemistry to demonstrate mycobacteria in Crohn's disease tissues. They examined 50 formalin-fixed, paraffin-embedded, tissues from 15 patients with Crohn's disease and found positive staining in areas of submucosal inflammation in 3 patients. One of the positive specimens was considered possibly to represent phagocytized red blood cells, and another possibly to be due to cross-reactions with non-acid fast organisms around an abscess, but the third could not be accounted for by any artifact. Control, non-Crohn's disease tissues, were not examined in this study. These authors acknowledged that their method, although successful in identifying organisms in positive control specimens, did not demonstrate mycobacteria in tissues from animals with experimentally induced intestinal mycobacterial infections. By conventional acid-fast staining, these tissues were either acid-fast negative or contained very few demonstrable organisms.

Kobayashi et al (148) failed to demonstrate mycobacteria in Crohn's disease tissues by immunohistochemical methods. They examined 67 specimens (from 30 patients with Crohn's disease), either fixed in formalin, periodate-lysine-paraformaldehyde, or not fixed, and reacted with anti-M. paratuberculosis, anti-M. tuberculosis, and monoclonal anti-LAM antisera. Although staining was observed in control tissues; e.g., lymph node from an AIDS patient, pulmonary tissue from two patients with mycobacterial infection, and liver from rats infected with M. kansasii, M. fortuitum, M. paratuberculosis, or spheroplasts of MAI serovar 26; no staining was observed in any of the Crohn's disease specimens. These authors indicated that their negative finding "eliminates mycobacteria as causing Crohn's disease in any conventional way." They assumed, however, that experimentally induced CWD forms in a constant state of reversion (as suggested by the presence of acid-fast and non-acid-fast forms) are immunologically identical to naturally occurring CWD forms and that their antisera which reacted with these experimental forms that it would also react with naturally occurring forms. Likewise, the detection of LAM in experimentally induced unstable CWD forms does not indicate that LAM exists in stable, natural CWD forms. At least one of their antisera used was previously shown to cross react with naturally occurring M. paratuberculosis spheroplasts (55). Their methods could detect large numbers of acid fast bacilli (e.g. tissues from AIDS patients or experimentally inoculated animals) as well as a few organisms as determined by acid-fast staining. Staining methods generally reveal 105 to 106 organisms per gram of tissue, i.e. reasonably large numbers. It has been shown that in some experimentally induced mycobacterial intestinal diseases, mycobacteria could not be detected by immunohistochemistry even though acid fast bacilli were known to be the cause of the lesion and could be cultivated. Demonstration of acid fast bacilli by peroxidase-anti-peroxidase immunohistochemistry was effective only in sections containing organisms visible by acid fast staining (Chiodini, R. J., J. A. Erickson, H. J. Van Kruiningen, W. R. Thayer, and J. A. Coutu. Gastroenterol. 90:1372, 1987; 282). It is unclear whether low concentrations of a Mycobacterium sp., e.g., 1000, 100, or even 10 per gram, are capable of producing disease in the gastrointestinal tract, but some documented cases of intestinal tuberculosis, paratuberculosis, and experimental studies suggest that very low numbers of organisms can cause a progressive disease.

The inability to detect mycobacteria or their antigens in tissue from patients with Crohn's disease is perhaps the most damaging evidence against their role as etiologic agents; however, if an agent associated with Crohn's disease could be easily demonstrated, it would have been found years ago. It is also unclear why one investigator failed to find any evidence of mycobacteria in 30 patients with Crohn's disease (148), another found evidence in one of 15 patients with Crohn's disease (282), and yet another found evidence in 34% of Crohn's disease specimens (Colemont, L. J. et al. Lancet 1988; 1:294-295). As Rubin and Pinner (237) have said about mycobacteria and sarcoidosis "the failure to find tubercle bacilli in the majority of cases ... is not a convincing argument against tuberculosis as an etiologic factor,
nor is the occasional tubercle bacillus which is found positive proof that tuberculosis is the cause".

If an infectious agent is present in Crohn's disease, it is either localized in very small foci or is scattered in small numbers, because immunohistochemical studies performed with polyclonal antisera should have identified any Mycobacterium spp. present. If mycobacteria are present, they are below the level demonstrable by the methods applied. Immunohistochemical techniques intensify staining of acid fast-positive sections in general, but their ability to detect mycobacteria in acid fast-negative tissue is questionable (R. J. Chiodini, J. A. Erickson, H. J. Van Kruiningen, W. R. Thayer, and J. A. Coutu. Gastroenterol. 90:1372, 1986). Such techniques need to be developed and standardized, and shown to work in tissues with low numbers of acid-fast bacilli, e.g., in culture-positive, acid-fast stain-negative cases of intestinal tuberculosis or paratuberculosis. Even more sensitive methods may need to be applied such as DNA hybridization; however, these techniques also have limitations. Specific genetic probes need to be obtained to prevent hybridization with complementary sequences of other microbes and eucaryotic cells. Liquid genomic DNA:DNA hybridization is probably not specific or sensitive enough to detect low numbers of organisms, and in situ hybridization is an extremely difficult technique which only a few laboratories are equipped to use. If the current etiologic theory is correct, additional problems become inherent. Can or will tissue DNA extractions liberate mycobacterial DNA since mycobacteria are so difficult to lyse? Application of standard tissue methods are not likely to liberate DNA from intact mycobacteria. Exposure of these organisms to chloroform (used in DNA extraction) generally makes the mycobacterial cell wall more ridgid and even more difficult to lyse. If the organisms exist in a CWD form, what is their stability after tissue death and what influences do exogenous eucaryotic DNases have on their genome? These factors need to be addressed and circumvented to adequately and convincingly conduct DNA hybridization studies. Again, acid fast-negative, culture-positive cases of intestinal tuberculosis or paratuberculosis would be the ideal model system for developing these techniques, and unless techniques are shown to be effective in these circumstances, the data generated will not be conclusive. These diseases provide a model of progressive granulomatous intestinal infection caused by very few, visually undemonstrable mycobacteria.

Of all the putative agents of Crohn's disease that have been isolated, until recently, none have been shown to be pathogenic for laboratory animals, humans, or specifically, the gastrointestinal tract. Because it has been assumed that its etiologic agent would show preference for gastrointestinal tissues, appropriate animals models have been sought to demonstrate the pathogenic potential of agents isolated from the tissues of Crohn's disease patients. In addition, such studies have been conducted as an indirect means of fulfilling Koch's postulates and thereby convincingly identify the etiology of Crohn's disease.

In 1984, using infant goats, Chiodini et al (49) described the first successful production of a granulomatous ileocolitis, or Crohn's disease-like infection, in experimental animals with a putative etiologic agent. Further, more detailed, studies with this goat animal model were later published in 1986 (281). Oral inoculation of goats with their putative agent, later identified as M. paratuberculosis, produced intestinal disease in approximately 5-6 months. The earliest lesions occurred within Peyer's patches of the ileum and consisted of non-caseous granulomatous clusters of epithelioid cells which often occurred in a mantle of lymphocytes between germinal centers and the muscularis mucosae; quite similar to the early lesions of Crohn's disease. Other features of the disease included tuberculoid granulomas without caseation, confluence of granulomas, ulcerations of the mucosa, and lymphocytic lymphangitis. Several animals had no demonstrable acid-fast bacilli although the bacillary organisms were isolated from all except controls. The authors concluded that the lesions produced in these animals were distinctly similar to those occurring in Crohn's disease.

Gitnick et al (98; personal communication) inoculated infant goats with a strain of M. chelonei subspecies abscessus isolated from a Crohn's disease patient, but failed to produce a granulomatous intestinal disease. These animals developed a transient diarrhea; intestinal lesions were limited to mild inflammation and colonic infiltration with polymorphonuclear cells. Others lesions seen in these animals were also present in controls and the findings were complicated by the presence of parasites. These investigators also inoculated infant goats with a strain of M. paratuberculosis isolated from a Crohn's disease patient, but again, failed to produce a granulomatous intestinal response; all animals remained normal during the 5 month observation period and lesions were not detected at necropsy.

The failure of these investigators to produce disease in ruminants with a human isolate of M. paratuberculosis is surprising because this organism is the etiologic agent of Johne's disease and there are no known non-pathogenic strains of M. paratuberculosis. In our original goat studies (48, 281), animals were fed viable organisms while Gitnick et al inoculated animals by stomach tube. Although the latter method would presumably be more precise than feeding, we also failed to produce disease by this method (unpublished data). Some physiologic change could occur by feeding M. paratuberculosis, which is essential to pathogenicity and which plays a role in the natural morbidity of this infection. Experimental infection studies with M. paratuberculosis in animals has always been performed by natural feeding (51); to the authors knowledge, no attempt has previously been made to infect animals with the use of a stomach tube.

Some recent data generated on natural M. paratuberculosis infection in animals has relevance to the animal model studies described above. McClure et al (172) described an epizootic of naturally occurring paratuberculosis in subhuman primates, stump-tailed macaques. Prior to this time, M. paratuberculosis was considered innocuous in primates, and this paper raised concerns regarding the potential public health implications of M. paratuberculosis infection. Several interesting features of paratuberculosis in subhuman primates were brought out by this article. Even though tissues containing large numbers of acid-fast bacilli were submitted to several laboratories for culture, none were unsuccessful in isolating the organism and reported either unculturable mycobacteria or unidentified acid-fast bacilli seen. The organism was not isolated until tissues were processed for M. paratuberculosis two years after the initial clinical case. Although the entire animal colony was thought to be infected on the basis of cultural data, no animal responded to tuberculin PPD or M. paratuberculosis antigens (johnin). Antibodies to M. paratuberculosis were present in all animals except those with clinical disease. Thus it was noted that neither cell mediated nor humoral immune responsiveness could be used to determine epidemiologic or diagnostic data. The authors concluded that either the M. paratuberculosis strain isolated had unique features that allowed it to infect subhuman primates or that the stump-tailed macaque was a susceptible host that had previously not been exposed to the organism. Finally, several severely ill animals were successfully treated with an experimental anti-mycobacterial agent, rifabutin (Adria Laboratories, Columbus; Farmatalia Carlo Erba, Milano) in combination with kanamycin, with subsequent disease remission. This was the first report of successful treatment of paratuberculosis and paved the way for drug trials in humans with Crohn's disease to be discussed later.

Czuprynski et al (63) described some the interaction of M. paratuberculosis with bovine macrophages and the experimental infection of gnotobiotic mice with M.
paratuberculosis. They found that intracellular growth of M. paratuberculosis could be restricted or enhanced by monocyte treatment with various cytokines. Intracellular replication of was restricted by crude interferon (IFN) or recombinant alpha interferon (rIFN-a) and enhanced growth resulted when monocytes were treated with a crude cytokine preparation obtained from an immunized animal. They concluded that production of this cytokine required the presence of both immune cells and M. paratuberculosis, suggesting that its production was dependent on specific lymphocytes (311). Experimental intragastric inoculation of euthymic and aghymic mice with M. paratuberculosis was also reported by these authors. In euthymic mice, a persistent low-level colonization occurred, while in athymic mice, progressive multiplication and consistent fecal shedding of M. paratuberculosis occurred. Organisms were recovered from the ileum of both animal groups, but were more abundant in athymic mice which also had demonstrable focal acid-fast clusters and occasional granulomas. M. paratuberculosis did not replicate in the intestinal lumen, even in the absence of competing microbes, but multiplication occurred in the mucosa. These findings are in agreement with others who demonstrated the inability of M. paratuberculosis to replicate outside the intestinal tissues and associated lymph nodes of its host (146, 184, 185).

Momotani et al (193) studied the mechanism of M. paratuberculosis infection in ligated ileal loops of calves. Within 5 h after inoculation, M. paratuberculosis had penetrated the intestinal lining and acid-fast bacilli could be visualized within subepithelial macrophages. By 20 h, greater than 50 bacilli per section were within subepithelial macrophages and in the supranuclear cytoplasm of M cells. Specific antisera seemed to enhance entry of M. paratuberculosis. It was concluded that M. paratuberculosis invades the intestines through the ileal M cells and that the subepithelial and intraepithelial macrophages secondarily phagocytize bacilli or bacterial debris which are expelled from the M cells.

Some recent animal experiments conducted at the University of Wisconsin in Madison yielded some unexpected results which also have relevance to animal models of Crohn's disease (H. A. Mokresh, S. Hurley, C. Czuprynski, and D. Butler, personal communication) . Newborn rabbits were orally inoculated with M. paratuberculosis and necropsied at various time periods to determine if intestinal lesions could be produced in these animals. Some developed a transient diarrhea and some developed a few intestinal granulomas. Interestingly, culture and prolonged incubation (11 to 15 months) of fecal and ileal tissue homogenates from some rabbits resulted in the growth of very small translucent colonies. The organisms stained poorly, and on electron microscopic examination, were found to be morphologically identical to the mycobacterial spheroplasts described previously in Crohn's disease (55). These CWD forms could not be subcultured. Should these results be repeated, they would represent the first successful in vivo transformation of mycobacteria into CWD forms. They may also represent an animal model for the physiologic and morphologic changes in M. paratuberculosis which may be occurring in the human intestine. These preliminary results have inspired further efforts which are currently in progress.

Perhaps as a follow-up of the suggestions first made by Dalziel (64) in 1913, and later by Golde and McGill (101) and Patterson and Allen (218), Morgan (194) recently published a theoretical paper comparing Crohn's disease and Johne's disease (paratuberculosis) and suggested that, based on previously reported experimental and epidemiologic data, these two diseases had similar etiologies. He noted the difficulties encountered in experimentally transmitting Johne's disease, not only to other cattle, but also to laboratory animals, as well as a host of other remarkable similarities. This author believed that the similarities, and disease histories,
were too remarkable to be coincidental.

It has been disputed that the experimental production of a granulomatous ileocolitis in goats with human isolates of M. paratuberculosis have little meaning and does not support an etiologic role of this agent in Crohn's disease (Graham, D. Y., D. C. Markesich, and H. H. Yoshimura, Letter, Dig. Dis. Sci. 33:251-252, 1988). Since the putative agent of Crohn's disease has been identified as M. paratuberculosis, the experimental infection is not Crohn's disease-like, but expectedly, is only Johne's disease. Such a conclusion, while taxonomically correct, should not be viewed as a simple distinction based on disease classification, and thereby separating two similar diseases. If Crohn's disease is not caused by M. paratuberculosis, then clearly the granulomatous ileocolitis produced in ruminants by human strains of M. paratuberculosis is Johne's disease. But, if Crohn's disease is caused by M. paratuberculosis, then the experimental infection produced in animals does represent Crohn's disease disease, even if the appropriate classification of the infection in animals is Johne's disease.

The isolation of a known animal pathogen from human patients with Crohn's disease has more implications to etiology than the isolation of a new or unknown species with no defined pathogenic characteristics. The fact that M. paratuberculosis is not an environmental organism and cannot replicate in the environment, readily penetrates and has a predilection for the gastrointestinal tract, produces a non-caseating granulomatous intestinal disease in animals, and has been isolated from the diseased tissues of patients with Crohn's disease, must at least raise issues of coincidence and suspicion amongst investigators.

Reports on the treatment of Crohn's disease with anti-mycobacterial agents are sporadic and generally not double-blinded nor well controlled with placebo treatment. Many are individual case reports (J. B. Warren, H. C. Rees, and T. M. Cox, Letter, 1986. New Engl. J. Med. 314:182; A. Picciotto, G. P. gesu, G. C. Schito, R. Testa, G. Varagona, and G. Celle, Letter, Lancet 1:536-537, 1988; C. Prantera, R. Argentieri, and R. Pangiarotti, Letter, Lancet 1:536, 1988) or involve few patients (216, 217, 274, 301). Antimycobacterial drugs have been selected randomly, based on their effectiveness in tuberculosis, and not efficacy with other mycobacterial species. Nevertheless, most studies have shown improvement, often long term, in Crohn's disease patients following administration of anti-mycobacterial chemotherapeutic agents. These long term effects, usually in severely ill patients, help rule out a placebo effect, but do not eliminate the possibility of spontaneous disease remission.

The only double-blinded study of antimycobacterial agents in Crohn's disease used rifampin and ethambutol with sulfasalazine and steroids versus sulfasalazine and steroids alone (245). Pursuing the possibility that M. kansasii was etiologically related to Crohn's disease, in 1984, these investigators reported on a 2-year randomized double-blind, crossover controlled trial with 27 patients. Thirteen patients were withdrawn before completion due to poor compliance, the need for surgical intervention, or adverse effects. Of the 14 patients that completed the trial, 4 required surgery, 5 were withdrawn because of poor compliance, and 5 were withdrawn because of drug side effects. Thus, their results were based on few subjects and shorter treatment periods than originally planned, Nevertheless, analysis of their data suggested that there was no significant difference in response to the anti-mycobacterial drugs as compared with sulfasalazine and steroids when expressed in terms of the Crohn's disease activity index (CDAI) or any other clinical indicator of disease activity, Additionally, there was no consistent pattern of change in the requirement for steroids in patients receiving anti-mycobacterial drugs. From their experience and data, these authors concluded that rifampin and ethambutol have no place in the treatment of Crohn's disease and that it was unlikely that M. kansasii was etiologically significant (245).

Recently there has been a surge of interest in the treatment of Crohn's disease with anti-mycobacterial agents, which undoubtedly, has been precipitated by the suggestions of M. paratuberculosis as the etiologic agent of this disease. Of the studies recently conducted or in progress, most have used rifabutin (Adria Laboratories, Columbus; Farmatalia Carlo Erba, Milan), a rifampin derivative, either alone or in combination with other drugs. The reasons for the interest in rifabutin as the anti-mycobacterial drug of choice is due in part to its high in vitro activity against M. paratuberculosis (unpublished data) and its successful use in the treatment of paratuberculosis in subhuman primates (172). Additionally, the manufacturer of rifabutin has been very supportive of its use as a chemotherapeutic agent in Crohn's disease. Nevertheless, the data accumulated to date has been poorly organized and difficult to interpret.

Basilisco et al (Basilisco, G., T. Ranzi, M. C. Campanini, L. Piodi, and P. A. Bianchi. 1988. Int Congress Gastroenterol. ; G. Basilisco, personal communication, 1988) reported on the use of rifabutin in a randomized double-blind trial in 24 patients with active Crohn's disease. Twelve patients received 300 mg rifabutin daily and 12 received placebo for 6 months. Nine patients (5 from the treated group and 4 from control) dropped out before completion. Of the remaining patients, 5 from the rifabutin-treated and 6 from the placebo-treated group improved transiently but in neither group was there any evidence of long term improvement in lesions or disease course. There was a high incidence of drug-related side effects, described as a flu-like syndrome, which precluded further studies.

Rutgeerts et al (Rutgeerts, P., G. Vantrappen, J. Van Isveldt, and K. Geboes. Gastroenterol. 94:A391, 1988) reported on the use of rifabutin and ethambutol of 6 months in 16 patients with Crohn's disease. In this trial, patients were evaluated based on pre-trial and follow-up ileo-colonoscopy and biopsies. Six patients withdrew from the trial due to the flu-like syndrome, and of the remaining 10, 7 completed the trial. No improvement was noted by endoscopy during the trial period and it was concluded that rifabutin/ethambutol treatment had no effect on the lesions of Crohn's disease. These investigators also noted the high occurrence of flu-like symptoms in patients on rifabutin and suggested that this high incidence might be due to an inherent disorder in Crohn's disease.

Mulder, C. J. J. (personal communication, 1988) initially performed an open trial with rifabutin and ethambutol of 6-month duration in 8 patients with Crohn's disease. Two patients improved clinically and are still doing well after 1-year. Of the remaining 6, all of whom declined the recommended surgery before entering the trial, no effect was noted in 3 and a temporary improvement was observed in 2, but surgical intervention was required on all. The eighth patient developed intestinal scarring, but otherwise improved with complete ulcer healing. These studies prompted the initiation of a small double-blind placebo trial in 15 patients; 7 receiving rifabutin and ethambutol, 8 receiving placebo. Although some improvement has been noted in the treated patients, the data is not very convincing. Two patients on placebo and 1 on drugs required surgery; laboratory parameters improved in 1 of the drug-treated patients as opposed to improvement in none and deterioration in 3 placebo-treated patients; the Harvey Bradshaw and Scope Crohn's disease index improved in 3, and deteriorated in 1 drug-treated patients versus improvement in none, and deterioration in 3 placebo-treated patients; and steroid dependence was reduced in 1 and increased in 1 drug-treated patients versus no reduction and
increased dependence in 2 placebo-treated patients. Although some minor effect may be occurring on drug-treated patients, the data presented is no very encouraging.

Thayer et al (Thayer, W. R., J. A. Coutu, R. J. Chiodini, and H. J. Van Kruiningen. Gastroenterol. 94:A458, 1988; unpublished data) used rifabutin in combination with
reptomycin in an open trial with 12 patients with Crohn's disease. Streptomycin (1-gram) was given intramuscularly 5-days a week for 2-4 months, and rifabutin was given orally at 300 mg/day for a minimum of 6-months or until drug withdrawal was elected. All patients were treated on a compassionate basis due to severe refractory Crohn's disease or because of extensive or repetitive fistularization and abscess formation. All patients have reportedly improved clinically, commonly with prednisolone withdrawal, healing of fistularization, and marked improvement in their CDAI. Of the 12 patients, 8 (66%) have completely withdrawn from steroids, 2 of 2 (100%) have withdrawn from 6-mercaptopurine or other drugs, 7 of 7 (100%) no longer have rectal bleeding, 4 (33%) have endoscopic healing of lesions, and 1 of 2 (50%) had radiographic improvement. These improvements were generally not noted until after at least 4-months duration of treatment and were most prominent after 6-months. Some patients failed to respond until after 6-months of treatment, thereby illustrating the need for long-term therapy in this chronic disease.

Hampson et al (Hampson, S. J., M. C. Parker, S. H. Saverymuttu, J. J. McFadden, and J. Hermon-Taylor. Gastroenterol. 94:A170, 1988;) treated 17 Crohn's disease patients with quadruple anti-mycobacterial chemotherapy. The antimicrobial agents used in combination were rifampin, ethambutol, isoniazid, and pyrazinamide with clofazimine replacing pyrazinamide in a few cases. Of the 17 patients, 12 (71%) had a statistically significant improvement in their CDAI and 9 of 10 (90%) had been completely withdrawn from steroids. At this date, these investigators have treated 20 patients with quadruple therapy, and after 9-months treatment, 11 of 20 (55%) are considered to be in disease remission. Based on 111 Indium scans, objective evidence of improvement after 1-year of treatment was found in 14 of 20 (70%) of patients (Hampson, S.A., personal communication, 1988).

The flu-like syndrome, which may or may not be associated with leukopenia, that is frequently observed in patients with Crohn's disease receiving rifampin or its derivatives is not understood. Other patients receiving rifampin or rifabutin, including patients with tuberculosis, leprosy, atypical mycobacteriosis, or acquired immune deficiency syndrome (AIDS), do not develop these symptoms; the flu-like syndrome appears unique to Crohn's disease patients. Although this syndrome was considered grounds for withdrawal from therapy in some studies, most consider that the severity is not sufficient to warrant drug withdrawal. Patients receiving steroids at the time rifabutin therapy is initiated fail to develop flu symptoms, i.e., steroids prevent the flu-loke syndrome. Thus, this condition is probably either related to drug toxicity or, considering the intervention by steroids, some form of immune phenomenon.

Although a mycobacterial etiology of Crohn's disease has been considered for well over 50 years, few studies have been conducted and little conclusive data are available on the effects, beneficial or not, of anti-mycobacterial chemotherapy. Often studies have been performed with little forethought and without supportive laboratory data.

Shaffer et al (245) failed to show any clear benefit of rifampin and ethambutol chemotherapy in Crohn's disease, however, several recent investigators chose and used rifabutin and ethambutol rather than evaluate other drug combinations. While rifampin and its derivatives show high in vitro activity against M. paratuberculosis, inethambutol does not (50) and should be considered a poor choice for dual therapy. Additionally, the use of rifabutin monotherapy would almost expectedly fail since the combination of rifampin and ethambutol is not effective and monotherapy is rarely, if ever, effective for treating mycobacterial diseases. The only studies, other than case reports, in which Crohn's disease patients have apparently shown improvement following anti-mycobacterial chemotherapy have been those employing rifabutin in combination with an aminoglycoside (which show high in vitro activity) or quadruple therapy. Unfortunately, most antibiotics to which M. paratuberculosis strains are susceptible in vitro are not available in oral preparations leading to more difficulty obtaining patient compliance and approval for experimental human use.

If Crohn's disease is caused by an organism similar to M. paratuberculosis, treatment schemes would need to follow current recommendations for other MOTT infections. General guidelines for the treatment of pulmonary mycobacterioses in non-immunocompromised patients include at least quadruple drug therapy and treatment durations of 2-3 years (6, 14, 129, 130, 277). Although the efficacy of treating pulmonary disease caused by MAI (closely related to M. paratuberculosis) has not been clear, current evidence suggests that treatment may be effective after prolonged periods (6, 14, 130, 277). In at least one recent study, clinical improvement of pulmonary MAI infection required 3.6 +/- 0.5 years of continuous chemotherapy (129). Such examples need to be evaluated in considering anti-mycobacterial chemotherapeutic regimes in the treatment of Crohn's disease.

In addition to the multiple drug regimes and prolonged therapy, whether or not anti-mycobacterial chemotherapy would be effective in Crohn's disease even if the etiology was mycobacterial also needs to be considered; chemotherapy is generally not effective in any known intestinal mycobacteriosis. In the treatment of hypertrophic ileocecal tuberculosis, surgical intervention is generally required since chemotherapeutic drugs alone are ineffective (3, 41). Some investigators consider that chemotherapy in hypertrophic intestinal tuberculosis is not necessary and should be provided, if at all, only as an adjunct to surgical intervention (41). Although paratuberculosis has been known as an intestinal mycobacterial disease since 1895, it has yet to be successfully treated despite the use of a wide range of antimicrobial agents (9, 51, 92, 93, 183, 230, 231). Even the prophylactic treatment of animals with antimycobacterial agents does not prevent experimental intestinal infection (231). Thus, the efficacy of treating intestinal mycobacterial diseases in general need to be considered.

Perhaps with the advent of new generations of drugs, and the recent successful treatment of intestinal paratuberculosis in subhuman primates with rifabutin in combination with kanamycin (172), effective chemotherapeutic drugs may become available. Nevertheless, data on the effects of anti-mycobacterial chemotherapy in Crohn's disease are needed and further evaluations of antimycobacterial drugs are warranted, particularly in view of the many case reports suggesting their efficacy. Future treatment studies, however, need to supported with solid laboratory data rather than just randomly selecting antibiotics. These studies should include not only in vitro susceptibility of organisms to individual antibiotics, but also antagonistic and synergistic effects of multiple drugs. Ideally, in vitro susceptibility profiles should be evaluated for treatment efficacy in an animal model system. Without background data on which to base human treatment schemes, such efforts will be greatly hindered. Lastly, one must consider that because anti-mycobacterial agents have broad activity, their effectiveness, in Crohn's disease, should it exist, is only supportive and not conclusive evidence of a mycobacterial etiology.

As has been noted since the first description of Crohn's disease in 1932, the similarities between Crohn's disease and mycobacteriosis are remarkable (59). Since that time, Crohn's disease and mycobacteria have been pushed apart so distantly that many of the common features have become obscure. Many consider that Crohn's disease received a too enthusiastic and uncritical acceptance as a unique disease entitity and that the diagnosis of primary intestinal mycobacteriosis was too lightly discarded. Primary hypertrophic intestinal tuberculosis does occur, and although early investigators thought that this disease was Crohn's disease, the two have been distinguished. In the western world, intestinal tuberculosis is generally misdiagnosed as Crohn's disease, and such cases are properly diagnosed only post-surgically (3, 39). On the other hand, in underdeveloped and those developing countries where tuberculosis is common, cases of granulomatous intestinal disease are generally diagnosed as tuberculosis. The similarities and dissimilarities of Crohn's disease and the mycobacterioses will be difficult to understand fully in the near future. A literature search from 1966 to September 1987 retrieves 7,661 reports on Crohn's disease or IBD and 31,429 on mycobacteria, tuberculosis, or leprosy. Comparisons must be made almost exclusively between Crohn's disease and tuberculosis or leprosy, because relevant data are limited to these human diseases. The similarities and differences are summarized below.

The pathology of Crohn's disease and intestinal tuberculosis, as well as other intestinal mycobacterioses, has been thoroughly reviewed and compared (128, 247, 269, 271, 296). While tuberculosis produces a characteristic and almost pathogonomic disease in most cases, in others, the disease is much less defined. Intestinal tuberculosis arising secondary to pulmonary disease is readily diagnosed by radiography of the thorax, presence of abundant acid-fast bacilli, and caseation necrosis. On the other hand, primary intestinal tuberculosis, i.e., intestinal infection without disease in other sites, may display non-specific pathologic features.

Three types of intestinal tuberculosis are recognized which depend, among other factors, on the virulence of the organism, resistance of the host, and the extent of illness. The ulcerative type is the most common and that generally associated with intestinal infection. It is always associated with pulmonary involvement. The ulcerohypertrophic type may occur as a result of pulmonary disease or as a primary infection, and results in ulcer healing with fibrosis and stenosis of the lumen. The hypertrophic form, which is rare and has been called pseudotuberculosis, is always a primary infection and is characterized by intense fibroblastic reactions in the submucosal and serosal layers of the bowel (3). Primary intestinal tuberculosis, particularly the hypertrophic form, is uncommon which probably explains why intestinal disease is difficult to produce experimentally with M. tuberculosis (39, 128). Hypertrophic tuberculosis may have a range of histopathological appearances from the frank presence to a complete absence of caseation necrosis, but its diagnosis is generally based on the demonstration of caseation necrosis, primarily in draining lymph nodes. Cultural efforts and the microscopic demonstration of acid-fast bacilli often times are negative.

Crohn and Yarnis (60) believed that the vast majority of hypertrophic tuberculosis cases were not tuberculosis, but rather examples of regional ileitis. Others have disagreed (40, 128, 131, 219, 269, 286), and if we accept M. tuberculosis as a strict pathogen, then hypertrophic intestinal tuberculosis without caseation necrosis or demonstrable acid fast bacilli does exist. Paustian and Bockus (219) established that at least one of their four criteria is needed to make a diagnosis of
intestinal tuberculosis: i) positive culture or guinea pig disease after inoculation; ii) microscopic demonstration of acid-fast bacilli in tissues; iii) presence of tubercles with caseation in diseased tissue; or iv) caseous granulomata in draining lymph nodes. All these criteria are seldom satisfied in hypertrophic tuberculosis, but at least one is generally accepted as sufficient for diagnosis (219). Acid-fast bacilli generally are not demonstrated and cultural attempts are positive in only a portion of cases. Adams and Holden (3) failed to demonstrate acid fast bacilli in 55% of patients with primary intestinal tuberculosis. Hoon et al (128) detected acid-fast bacilli in only 33% of 58 cases examined. Stains for acid-fast bacilli in or around tubercular fistulae are always negative. Shah (247) examined 20 cases of hypertrophic intestinal tuberculosis and successfully isolated M. tuberculosis in only 7 (35%) cases. Wig et al (296) were successful in cultivating organisms in only 26 (35%) of the 69 cases of hypertrophic tuberculosis they examined. In other cases, diagnosis was made based on the presence of caseation necrosis of the diseased tissues. Such necrosis, however, is generally absent in intestinal tissues and is only observed in the draining lymph nodes. Thus, the importance of lymph node histopathology in the diagnosis of primary hypertrophic tuberculosis of the intestine has been stressed and that the lack of caseation necrosis does not exclude the diagnosis of tuberculosis. Caseation necrosis of draining lymph nodes may be present in as few as 24.4% of culture-positive cases of intestinal tuberculosis (62). Among culture-positive cases of hypertrophic tuberculosis, Wig et al (296) found 10 cases in which there was no caseation and the lesions were limited to non-specific inflammation. Thus, the ability to demonstrate acid-fast bacilli in cases of hypertrophic tuberculosis of the intestine is poor, as is the ability to culture M. tuberculosis, and in some cases, caseation necrosis is absent. There are no documented cases of intestinal tuberculosis in which acid fast bacilli were not demonstrable, cultures for M. tuberculosis were negative, and no caseation necrosis was seen. Such cases most likely would be diagnosed as Crohn's disease, if pulmonary radiographs were normal.

Every clinical, radiologic, endoscopic, and pathologic feature of Crohn's disease may occur in primary intestinal tuberculosis or some other mycobacterioses, and they are indistinguishable (Table 5, Table 6, Table 7). Both occur most frequently in the ileocecal region, and both may occur anywhere in the gastrointestinal tract from mouth to anus. In the United States where ileocecal tuberculosis is rare, such cases are generally diagnosed only after surgical resection for Crohn's disease (3). When the features of these two diseases are compared, the only distinguishing criteria are the presence of caseating granulomas and acid-fast bacilli in tuberculosis. Thus, the absence of caseation necrosis and failure to isolate or demonstrate mycobacteria are the chief if not sole criteria for the diagnosis of Crohn's disease. As discussed, these are not reliable criteria. Taylor (271), in his study of intestinal tuberculosis and Crohn's disease, concluded that "it is impossible on the basis of clinical features or morbid anatomy to distinguish between these two conditions". Cattel and Mosely (41) shared this view and stated that ileocecal tuberculosis and Crohn's disease "may be virtually impossible" to distinguish. Even Crohn himself, in a discussion of the paper of Watson et al (287), conceded that "any pathologist would have difficulty in differentiating the pathology of so called pseudotuberculosis, of defining histologically sarcoidosis, ileitis, or ileojejunitis". Without doubt, certain cases of hypertrophic tuberculosis of the intestine are difficult, if not impossible, to differentiate pathologically from Crohn's disease. Some investigators have compared intestinal tuberculosis and Crohn's disease, and have provided detailed pathological descriptions that allow their differentiation (269). These reports, however, did not describe hypertrophic tuberculosis but dealt primarily with secondary intestinal disease or the ulcerohypertrophic variety.

Crohn's disease and mycobacterioses not only share the features of primary intestinal disease, but of extra-intestinal manifestations as well. In Crohn's disease, arthritis, iritis, erythema nodosum, and amyloidosis are occasionally encountered and are considered to be important extra-intestinal manifestations (28, 66, 124, 142, 152, 212, 300). Arthritis is a well-known complication of mycobacterial infections (126), and in recent years, it has been shown that arthritis can be produced by mycobacterial antigens alone (127, 280). Erythema nodosum has its counterpart in leprosy, a condition known as erythema nodosum leprosum (117, 288). Amyloidosis may occur in intestinal tuberculosis (62, 128), leprosy (70, 285) and paratuberculosis (Johne's disease) of animals (32, 51, 199). Ocular lesions occur in leprosy (264) and are occasionally encountered in paratuberculous animals (199).

Comparisons of Crohn's disease pathology have been made almost exclusively with tuberculosis, yet M. tuberculosis most likely is not the etiologic agent of Crohn's disease. The major distinguishing feature between Crohn's disease and primary intestinal tuberculosis is the presence of caseation necrosis and pulmonary lesions, features of disease produced by the M. tuberculosis complex, but not necessarily other mycobacteria. Therefore, if Crohn's disease is caused by some other Mycobacterium spp., caseation necrosis need not be present. In addition, M. tuberculosis intestinal infections are not readily produced experimentally, suggesting that this is not a preferred site of the organism. In contrast, M. paratuberculosis, a more likely candidate as the etiologic agent, has a strict preference for the gastrointestinal tract and does not produce caseation necrosis.

The population epidemiology of Crohn's disease and the mycobacterioses are not readily comparable because of the manner in which these studies have been conducted. Whereas the epidemiology of tuberculosis is well defined and based on total populations studies, that of Crohn's disease is not. There are no methods for population surveillance (such as PPD reactivity), and prevalence/incidence data are determined regionally through hospital records. Additionally, Crohn's disease population epidemiology is hampered by a long lapse between onset of clinical symptoms and diagnosis and an unequal precision in the use of diagnostic criteria in different study centers. In a proportion of studies it has been determined that approximately 20% of the study group are misclassified and do not have Crohn's disease (35). Thus, epidemiology will be addressed breifly.

Crohn's disease occurs with its highest incidence in the United States, the United Kingdom, and Scandinavia. It is less frequent in Central Europe and rarely is reported in Africa, Asia, and South America. The disease is seldom reported in underdeveloped or developing countries (35, 36). In the United States, the incidence is somewhere between 3.1 to 13.5 per 100,000 population, and is between 0.3 to 7.3 in other countries where the disease is reported (35, 36). Reports are conflicting, but the incidence of Crohn's disease in the United States and in other countries has been increasing, particularly in certain regions (35, 36, 91, 208, 262). Generally, the prevalence of disease appears to have stabilized in most countries. In contrast, tuberculosis (and leprosy) occur with highest frequency in those areas where Crohn's disease is rarely seen, and with low frequency where Crohn's disease is most frequent.

The incidence of tuberculosis in the United States has been decreasing for the last few decades, but has in recent years begun to increase. Some individuals have suggested that the apparent increase of Crohn's disease in the western world is related to the decreasing incidence of tuberculosis, because infection or immunization with one Mycobacterium species provides protection against infection with another species. For example, BCG and other mycobacterial antigens provide some cross-protection against leprosy and other mycobacterial diseases (160, 210, 250, 265); M. avium vaccination protects cattle from M. paratuberculosis infection (51). The American black population had an increased incidence of Crohn's disease, at about the same time the reported tuberculosis rate was decreasing (44). The prevalence of Crohn's disease has now appeared to stabilize in the United States, again corresponding with the current rise in M. tuberculosis infection. Although the protection afforded by endemic tuberculosis may be less common in the western world, most likely, the mis-classification of disease accounts for the geographic distribution of Crohn's disease. Nevertheless, from the period 1960 through 1980, the prevalence of tuberculosis decreased 55% in the United States (163) and Crohn's disease increased 38% and 61% in Baltimore and Amstead County, Minnesota, respectively (35).

In Table 8, epidemiologic data about Crohn's disease, ileocecal tuberculosis, and paratuberculosis are compared. In women who are of English or northern European descent, the incidence rate of Crohn's disease is 30% greater in age-matched males (35). The age incidence of Crohn's disease shows a bimodal distribution. The primary incidence mode occurs at ages 15 to 25, followed by a second mode at ages 55 to 60 (35). Of the 121 Crohn's disease patients studied by Schoffield (243), 84% were under the age of 40 and 75% were females. While pulmonary tuberculosis has a greater frequency in males, primary ileocecal tuberculosis is predominant in females, approximately 70% of cases (247, 296). The maximum age incidence of intestinal tuberculosis is also 15 to 24 years (296), with 65-85% of patients being under the age of 40 (247). If we assume that Crohn's disease and human intestinal tuberculosis occur at the prime of life (15-25 years of age), then a similar maturity incidence occurs in animals with paratuberculosis. The maximal age incidence of paratuberculosis in cattle is 3 to 5 years, during their prime of life and period of
maximum productivity (51). Since animals in the cattle industry are primarily female, a preponderance for disease in females cannot be assessed. The only feature that is not almost identical between Crohn's disease and intestinal tuberculosis is the presence of a secondary age incidence mode, which is not invariable (35). Insufficient cases of primary intestinal tuberculosis have been examined to determine if a secondary mode exists. Because a secondary age mode is well documented in pulmonary tuberculosis, it is likely that such does occurs in the ileocecal disease as well. In pulmonary tuberculosis, this secondary age incidence mode arises as a result of degeneration of a Ghon lesion acquired earlier in life (196). Perhaps a Ghon-type lesion occurs in the gastrointestinal tract.

There is a known familial association of Crohn's disease (84, 144, 157, 170, 259, 290) which suggests a genetically-linked increased susceptibility to the disease, or alternatively, a common exposure to an etiologic agent. There is a low incidence of Crohn's disease in married adults, but these rare occurrences have yet to be explained (228, 295; Bennett, R., P. H. Rubin, and D. H. Present. Gastroenterol. 94:A611, 1988). A genetic link, as assessed by HLA-typing, has not been found (88), but genetic predisposition is likely. There is a 30 times greater rate of Crohn's disease in siblings and 13 times greater incidence in first degree relatives (84, 85). Such a familial association, the occurrence of Crohn's disease in siblings and mono- and di-zygotic twins (including those living apart since early childhood), and the rarity of Crohn's disease in half-siblings (21, 30, 85, 145, 203), indicates a genetic susceptibility or predisposition occurring as a recessive trait.

Morgan (194) has proposed an alternative explanation based on the epidemiology of M. paratuberculosis infection in animals. In this disease, animals are infected with M. paratuberculosis during early childhood (before 30-days of age) but disease
becomes manifested later in adult life. An age-dependent resistance develops such that adult animals not exposed to the agent during early life rarely become infected, even experimentally. Thus, Morgan postulated that early exposure to an infectious agent (M. paratuberculosis) would account for the occurrence of Crohn's disease in siblings and mono- and di-zygotic twins, and the rarity of Crohn's disease in half-siblings. He felt that the case for early infection was particularly supported by the disease occurrence in twins separated since childhood. Since paratuberculosis is rarely transmitted to adult animals due to age-dependent resistance, a low incidence in adult married couples would be expected. Morgan proposed a time-space clustering study of Crohn's disease patients during their first 5-years of life to address these issues.

As would be expected in a chronic granulomatous disease, there is no consistent humoral immune dysfunction in Crohn's disease. Although a few reports have described an intrinsic B cell defect and dysfunction (165, 251), these have not been found by other investigators (79, 165). There may be an increased number of IgM-bearing cells in the intestinal mucosa (165). Perhaps the only humoral immune finding in Crohn's disease is an increased number of spontaneous Ig secreting cells and a decrease in responsiveness to B cell mitogens during active disease (165), suggesting an in vivo polyclonal B cell activation.

A high proportion of Crohn's disease patients have auto-antibodies against gastrointestinal tissue and other self antigens (8, 260). Although these anti-colon antibodies may arise due to cross-reactivity with E. coli 014 K1 antigens (260), the documentation is rather weak. Circulating immune complexes are also observed in Crohn's disease (67, 99, 153). Although they are not consistently detected, it is now known that their presence correlates with episodes of clinical disease. Both of
these manifestations are probably secondary to the disease state even though they may have clinical relevance. Auto-antibodies are commonly found in mycobacterial infections and are thought to arise as a result of the potent mitogenic and adjuvant activity of mycobacteria. Auto-antibodies have been investigated most thoroughly in leprosy where anti-skin, anti-DNA, anti-neuron, and a host of other self-antibodies arise as secondary phenomena of infection (81, 90). In leprosy primarily, but also in tuberculosis (65, 169, 257), circulating immune complexes frequently occur and are commonly associated with episodes of erythema nodosum leprosum (89, 90, 207, 229). Both the presence of low level auto-antibodies and circulating immune complexes are common manifestations of chronic disease in general.

Studies on cell-mediated immune function in Crohn's disease are conflicting. Although many studies have shown dysfunction or intrinsic cell defects (15, 73, 75, 80, 209, 251, 268, 303), others have found no abnormalities (23, 29, 79, 86, 134, 150, 164). Decreased cytotoxicity has been observed in Crohn's disease (15, 16), as well as in ulcerative colitis, but this effect is believed to be caused by an increased number of immature monocytes in the peripheral blood as a result of rapid turnover rate. Increased or defective suppressor cell activity has also been observed in Crohn's disease (75, 87, 103), and although reports conflict (134, 221), increased suppressor cell activity is common in chronic disease. The only consistent finding is a reduced number of T cells in peripheral blood (213), but in general, there are no imbalances or dysfunctions of helper or suppresser T cells, and no alterations in T cell immunoregulation or function in the peripheral blood or in the intestinal mucosa of Crohn's disease patients (29, 82, 100, 173, 214, 221, 309). The failure to detect consistently a humoral or cell-mediated immune dysfunction may mean only that none yet has been demonstrated. Crohn's disease patients elicit an abnormal and exaggerated immune response in the gastrointestinal tract characterized by a DTH reaction. Because so little is known and understood about the immunology of
Crohn's disease, comparisons cannot be made with the immunology of other mycobacterioses (45, 46, 51, 119, 158, 202).

In many respects, Hippocrates was correct when he said "Diarrhoea attacking a person with phthisis is a mortal symptom" (4). While people with pulmonary tuberculosis (phthisis) occasionally did improve, secondary infection of the gastrointestinal tract was always fatal. This was also true for patients with primary ileocecal tuberculosis; advances in abdominal surgery provided relief rather than the advent of chemotherapeutic agents. Primary ileocecal tuberculosis, especially the hypertrophic type, is not responsive to drug therapy alone and surgical resection of the bowel is required (20, 41). Chemotherapy is only an adjunct to surgical intervention, and is often not necessary (3, 41).

Chemotherapeutic treatment of Crohn's disease generally involves the use of prednisolone and/or sulphasalazine (236), with surgical intervention needed in 60 to 80% of patients (102). The mechanism of action of prednisolone is known, that of sulfasalazine is less clear. Sulfasalazine is cleaved by colonic bacteria into sulphapyridine and 5-aminosalicylic acid (12), which are believed to be the active products. A variety of other drugs have been evaluated, and are occasionally used, but currently, no chemotherapeutic drug or regime has ever provided a cure for Crohn's disease. Treatment and disease management is supportive.

The current data on the use of anti-mycobacterial chemotherapy in Crohn's disease has been previously discussed. Some data, particularly case reports, strongly suggest a beneficial effect, but larger studies have not been as encouraging. Sufficient data, however, are not available to precisely define the effects of these agents on Crohn's disease. Nevertheless, an understanding of the use of chemotherapy in mycobacterioses is essential in order to appreciate the
potential application of this form of therapy.

Any discussion of chemotherapeutics in Crohn's disease must consider the placebo effect and spontaneous clinical remission. Several large studies (166, 267) have shown that 25 to 40% of patients receiving placebo improve enough during the first 3 to 4 months of treatment to be considered to have gone into clinical remission. About 20% of placebo-treated patients remain well after 1 year and 10% after 2 years. Such remissions may even be accompanied by radiographic improvement (188). Drug toxicity occurs in 6 to 8% of patients on placebo medication. The effect of placebo on maintaining clinical remission is even more striking. Of 20 patients achieving remission while on placebo (267), 15 (75%) remained well for at least 1 year. Of 11 patients followed for a second year, 7 (63%) remained in remission. Thus, any data presented on the treatment of Crohn's disease which is not performed in a double-blinded placebo fashion and/or is performed only over a short period of time, must be interpreted with the knowledge that 42% of ill patients may get better without any specific therapy during the first 3 to 4 months (166, 188, 267). The placebo effect may be reduced after 1 to 2 years, after which continued improvement is evident in only 20 and 10% of patients, respectively.

The use of anti-mycobacterial therapy in mycobacterial disease is a well established therapeutic approach, but it is not always effective, particularly in certain diseases or disease-types. Chemotherapeutic drugs alone are ineffective in the treatment of hypertrophic ileocecal tuberculosis. This disease state requires intestinal resection (as in Crohn's disease), with antimicrobial agents provided only as adjunct therapy. Paratuberculosis, a well recognized intestinal mycobacterial disease of ruminants, has yet to be successfully treated despite the use of a wide range of antimicrobial agents (51). Prophylactic treatment of animals with antimycobacterial agents does not even prevent experimental intestinal infection (231). Therefore, it must be appreciated that although anti-mycobacterial agents are effective in classical diseases such as tuberculosis and leprosy, they are not effective in any known intestinal mycobacterioses. These drugs would probably have limited efficacy in Crohn's disease even if it was caused by a Mycobacterium species.

The use of steroids in Crohn's disease patients has been a major argument against a mycobacterial etiology. Steroids and other immunosuppressive therapy, is considered contraindicated in pulmonary tuberculosis, causing an exacerbation of the disease. However, the detrimental effects of immunosuppressive drugs on mycobacterial infections are not as pronounced as believed. Steroids in combination with antimicrobial agents have been used for treatment in leprosy (133, 227, 246) and in tuberculosis and other mycobacterial infections (125, 204). Retrospective studies have now established that corticosteroids do not cause re-activation of pulmonary tuberculosis (112) as case reports had long suggested especially if only small numbers of bacilli are present. Re-activation of disease due to steroid therapy is more likely caused by the chronic condition that mandated steroid therapy than the metabolic effects of the steroid (112). Studies in cattle with paratuberculosis have shown that massive corticosteroid administration does not significantly influence the clinical manifestations or outcome of the disease, although it was expected to (7). Treatment of experimental M. paratuberculosis infection in rabbits with methotrexate, a powerful immunosuppressive drug, resulted in clinical improvement even though the bacillary load increased (186). In Crohn's disease, as in tuberculosis and leprosy, steroids are used to provide clinical relief, but neither disease can be cured by such treatment. Although disease remission occasionally occurs in Crohn's disease patients receiving corticosteroids, it is difficult to determine if this remission is due to the steroid therapy, occurs spontaneously (placebo effect), or reflects a masking of the disease as occasionally observed in leprosy (227).

Non-steroidal anti-inflammatory agents are known to activate quiescent Crohn's disease (140) and induce intestinal inflammation in other types of patients as well (24). Therefore, their use is contraindicated. Non-steroidal anti-inflammatory drugs are also known to activate quiescent pulmonary tuberculosis (275) and are also contraindicated in mycobacterioses.

In summary, chemotherapeutic schemes offer several similarities between the mycobacterioses and Crohn's disease: i. anti-mycobacterial agents appear effective in only a portion of Crohn's disease patients as they are in intestinal mycobacterioses; ii. corticosteroids offer clinical improvement in Crohn's disease, leprosy, and some cases of tuberculosis and other mycobacterioses; and iii. non-steroidal anti-inflammatory agents activate quiescent Crohn's disease and tuberculosis. Despite these similarities, the efficacy and appropriateness of antimycobacterial chemotherapy in Crohn's disease remains to be evaluated.

In the preceding pages, experimental and comparative data have been presented on the association of mycobacteria and Crohn's disease. A large portion of this information is either preliminary or in abstract form and must be interpreted with caution. While no firm evidence clearly identifies mycobacteria as an etiologic agent, the notion is supported by suggestive and circumstantial data, and remarkable similarities to other known mycobacterial diseases. A consensus could probably be reached on the notion that if the etiology of Crohn's disease is microbial in origin, then it is most likely mycobacterial.

All major texts on gastroenterology and mycobacteriology make reference to the possible mycobacterial etiology of Crohn's disease. This is surprising, particularly in older texts, since the data which suggested such an association was sparse when these texts were written (Table 9). Crohn et al (59) dismissed the notion of a mycobacterial etiology with their description of Crohn's disease in 1932. It was 20 years later that Van Patter presented his doctoral thesis seeking to associate mycobacteria and Crohn's disease. These efforts were never formally published, therefore presumably this information was not generally known. Twenty-six years later Burhnam et al (34) published their data on M. kansasii and Crohn's disease. Since Van Patter's work was not referenced by any text or article during this period, the first concerted effort to associate mycobacteria and Crohn's disease was in 1978, 46 years after Crohn dissociated the disease from mycobacteria. Recently a concerted effort has again been made to investigate the possible association of mycobacteria and Crohn's disease but the fact that text written prior to 1984 considered mycobacteria as a possible etiologic agent of Crohn's disease indicates that the medical community has never dismissed this notion.

The relationship between Crohn's disease and mycobacteria is an old idea that has never been thoroughly investigated. Data are just now becoming available through active research efforts. The notion of a mycobacterial etiology of Crohn's disease should be viewed with skepticism and criticism, but the level of controversy surrounding this issue is exaggerated considering the data which was available prior to this latest surge of effort (Table 9).

Perhaps the biggest error in the study of Crohn's disease is the assumption that Crohn's disease is a single disease entity. The clinical and pathological criteria of Crohn's disease are not specific enough to ensure precise diagnoses. No unique features identify Crohn's disease except the inability to diagnose or associate the signs and symptoms with another disease. There is such great variability between patients that Crohn's disease probably reflects a variety of diseases grouped into one. The histologic hallmark of this disease, i.e., non-caseating granulomas, is found in only 40 to 60% of cases (198, 242), or even fewer (132). Based on
epidemiologic data it has been estimated that at least 20% of Crohn's disease diagnoses are mis-classifications (35), but this figure is probably conservative. Adding to the confusion is the grouping of Crohn's disease along with ulcerative colitis as IBD. Such data are near impossible to interpret as two distinct disease entities. If what we now know as Crohn's disease is not a single disease entity, statistically significant data cannot be achieved. Investigations need to be conducted with well characterized patient populations and pathological material to limit the possible effects of mis-classification and grouping together of several disease entities. Even if Crohn's disease is found to be caused by a mycobacterial agent, the disease would probably be confirmed in only a proportion of the patients we now consider to have this entity.

If Crohn's disease has a mycobacterial etiology, the most likely agent would be M. paratuberculosis. This organism is unique among the mycobacteria because the gastrointestinal tract is the only environment in which it can replicate in vivo. As M. leprae and M. ulcerans favor the skin, M. paratuberculosis favors the intestines and associated lymph nodes. It is incapable of survival (replication) in a variety of environmental materials, and has never been found in any environmental source not associated with disease. By-products of its own environ are toxic to it; feces are bacteriostatic and urine is bactericidal. Because it is unable to live in the environment, it must be considered a strict pathogen (51). It is clearly suited to cause a chronic granulomatous ileocolitis in ruminants an\ sub-human primates, and perhaps, even humans. It's disease remains incurable.

The question to the role of mycobacteria as etiologic agents in Crohn's disease is likely to remain unanswered for years to come. Such an agent would need to (i) exist in an unculturable fashion (perhaps as a spheroplast); (ii) occur at concentrations below the microscopic detection level of current technology ( <105 per gram of tissue); (iii) fail to elicit a strong humoral immune response as in polar tuberculoid leprosy; and (iv) remain at low concentrations even when steroid therapy is administered. Perhaps it would even fail to elicit a DTH skin reaction as in hypertrophic ileocecal tuberculosis; such a disease syndrome, if it exists, would be difficult to detect. The only circumstance in which these conditions could occur would be the development of local DTH reactions (as in tuberculoid leprosy) within the intestinal tissues. A competent immune system capable of hindering bacterial proliferation at the macrophage level could result in low bacillary loads. An unmetabolizable product that could induce DTH and occur either as part of the organism, e.g. certain components of the mycobacterial cell wall, or as a by-product of metabolism or degradation could produce a progressive chronic disease. Mycobacterial cell walls inoculated into the lungs of mice produce progressive hypersensitivity pneumonitis in the absence of viable organisms (18, 159, 179, 256). The M. paratuberculosis vaccine used in cattle, results in a large granuloma which lasts for the lifetime of the animal. This vaccine, accidentally injected into humans, results in progressive granulomatous inflammation necessitating surgical amputation of the injection site (51). This same vaccine, given to sub-human primates resulted in a severe progressive and disseminated disease suggesting the presence of viable organisms (H. M. McClure, personal communication). Cultures of the vaccine were negative, as were the animal tissues at autopsy. Such potent immune modifiers could conceivably produce the syndrome known as Crohn's disease.

Future research direction is clear. Efforts need to be concentrated on techniques for demonstrating low concentrations of mycobacteria in tissues. Two possible routes exist. The use of specific genetic probes or the more readily available monoclonal antibodies. A variety of mycobacterial monoclonal antibodies react with genus-specific antigens and many against cytoplasmic components. These latter monoclonal antibodies should detect CWD-forms; although they would not
identify the particular species. Important information about the role of mycobacteria and Crohn's disease could be gained. Species specific genetic probes, particularly against M. paratuberculosis, need to be used, but the genus-specific probes could provide the framework for future efforts. Microbiologic efforts need to concentrate on improved cultivation and isolation techniques, transformation of CWD-forms into classical bacillary forms, and methods to precisely identify these CWD-forms.

Since Crohn's disease is a DTH-mediated disease, efforts need to concentrate on examining the CMI and DTH in Crohn's disease patients. Such studies need to be performed not only on peripheral blood cells, but intestinal mucosal cells as well. Unfortunately species-specific antigens are not available and are not likely to be forthcoming in the near future. Mycobacterial antigens have been exhaustively examined and many monoclonal antibodies developed, yet the species-specific antigens detected as far and few between (66). However, effort direction can be obtained by using non-specific antigens.

Areas of research of less importance include antigen purification, humoral immunity, and animal model studies. Sufficient data have been obtained to document that Crohn's disease patients do not have a consistently demonstrable antibody response to mycobacteria. Although this could very well be related to nonspecific mycobacterial antigens and cross-reactions with environmental mycobacteria, highly purified immunogenic antigens are not now available and probably will not be for many years. Studies seeking seeking specific antigens are too time consuming to warrant much effort at this time. If mycobacteria are established as the cause of Crohn's disease, then purified antigens for use in diagnostics would be an appropriate effort. Likewise, animal model studies are of limited value at this time. If mycobacteria are found not to be the cause of Crohn's disease, production of a granulomatous ileocolitis resembling Crohn's disease in goats or mice following
inoculation of mycobacteria has little significance as an animal model. Such models need to be developed only after the cause is established. Some investigators seeking animal models to prove a mycobacterial etiology consider that the pathologic disease must be identical to that found in Crohn's disease (H. J. Van Kruiningen, Dig. Dis. Sci. 33:251-252; 1988). While this situation would be ideal, it is unrealistic. Mycobacterial diseases are essentially immunologically-mediated disorders, therefore, each species responds immunologically differently and identical pathologic diseases would not be expected. For example, M. paratuberculosis infection in cattle does not produce caseation necrosis, but 25% of goats develop caseating granulomas in response to infection with M. paratuberculosis (51). Humans are likely to respond differently. Microbiologic efforts to isolate mycobacteria must also be considered an area of less priority since such efforts have not been very productive in the past. If Crohn's disease is caused by a Mycobacterium spp., perhaps in a spheroplast form, current techniques are inadequate to insure consistent isolation and/or propagation. New methods need to be developed.

It is highly unlikely that mycobacteria cause all cases of Crohn's disease, but available data suggest strongly that they do cause some cases. The mycobacterial etiology theory of Crohn's disease remains alive. Despite the negative data generated, the similarities of Crohn's disease and the mycobacterioses are too remarkable to dismiss as coincidental. Perhaps with the new wave of interest in this old idea, an answer to this persistent question will soon be forthcoming.

Regardless of the outcome of current studies, the pathologic findings, familial occurrences, extra-intestinal manifestations, ectopic sites of disease often occurring concurrently with intestinal infection, and the recurrence of disease at resection margins, all suggest an infectious etiology. Efforts to find that agent will undoubtedly continue.

The support provided by research grants from the National Institute of Allergy and Infectious Disease, the National Institutes of Health, and the National Foundation for Ileitis and Colitis, Inc. is acknowledged.

253. Sigurdsson, B. 1946. A specific antigen recovered from tissue infected with M. paratuberculosis (Johne's bacillus). II. Studies on the nature of the
antigen and on methods for demasking it. J. Immunol. 53:127-135.

254. Sigurdsson, B. 1947. A specific antigen recovered from tissue infected with M. paratuberculosis. III. Further studies on the nature of the antigen and on
methods for demasking it. J. Immunol. 55:131-139.

279. United States Departmment of Agriculture. 1974. Laboratory methods in veterinary mycobacteriology for the isolation and identification of mycobacteriology. Mycobacteriology Unit, National Veterinary Services
Laboratory, APHIS, Ames, IA.

*Number of isolates per number of patients. Percent isolation provided in parentheses. Bracketed results indicate that mycobacterial identity not
confirmed.bnumber of patients includes controlscNot known.

aReferences cited in textbFemales comprise major population in domestic livestock such that predonderance cannot be determinedcAge designation beyond lifespan of domestic livestock. Paratuberculosis rarely occurs in older animalsdNormal agricultural life of livestock not long enough to determine.